Accepted Article This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/tpj.14299 This article is protected by copyright. All rights reserved. ArticleType : Original Article Hybrid sequencing reveals insight into heat sensing and signaling of bread wheat Xiaoming Wang 1 , Siyuan Chen 2,3 , Xue Shi 1 , Danni Liu 4 , Peng Zhao 1 , Yunze Lu 1 , Yanbing Cheng 4 , Zhenshan Liu 1 , Xiaojun Nie 1 , Weining Song 1 , Qixin Sun 5,1 , Shengbao Xu 1* , Chuang Ma 2,3* 1 State Key Laboratory of Crop Stress Biology for Arid Areas, College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi, China. 2 State Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China. 3 Center of Bioinformatics, College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China. 4 Frasergen, Wuhan East Lake High-tech Zone, Wuhan 430075, China. 5 Department of Plant Genetics & Breeding, China Agricultural University, Yuanmingyuan Xi Road No. 2, Haidian District, Beijing 100193, China. *Correspondence: Shengbao Xu, [email protected]and Chuang Ma, [email protected]Running title Heat sensing, signaling and adaptations of wheat Keywords heat sensing and signaling, early heat stress, hybrid sequencing, spatio-temporal transcriptome, transcriptional regulation, alternative splicing (AS) regulation, wheat (Triticum aestivum L.).
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This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/tpj.14299
This article is protected by copyright. All rights reserved.
ArticleType : Original Article
Hybrid sequencing reveals insight into heat sensing and signaling of bread
wheat
Xiaoming Wang1, Siyuan Chen
2,3, Xue Shi
1, Danni Liu
4, Peng Zhao
1, Yunze Lu
1, Yanbing
Cheng4, Zhenshan Liu
1, Xiaojun Nie
1, Weining Song
1, Qixin Sun
5,1, Shengbao Xu
1*, Chuang
Ma2,3*
1State Key Laboratory of Crop Stress Biology for Arid Areas, College of Agronomy,
auto.jsp?dir=/download_files/Genes), respectively. Isoforms with CPC score ≥ 0.44 were also
considered to have protein-coding capacity. The threshold of the CPC score (0.44) was
optimized by applying a 10-fold cross-validation approach, resulting to the generation of a
balanced sensitivity and specificity (0.91) for CPAT.
Acknowledgments
We thank IWGSC for providing the pre-publication reference sequences and genome
annotation of the bread wheat variety Chinese Spring. We also thank Dr. Kellye Eversole
(Eversole Associates, United States) for critical reading of the manuscript.
This work has been supported by the National Natural Science Foundation of China
(31570371 [Ma]), (31501380 [Wang]), the Youth 1000-Talent Program of China (Ma), the
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Hundred Talents Program of Shaanxi Province of China (Ma), the Fund of Northwest A&F
University (Ma, and Xu), and the Basic Research Project for Natural Science of Shanxi
Province (2016JQ3023 [Shi]).
Conflict of Interest Statement
The authors declare no conflict of interest.
Accession numbers
The data reported in this article have been deposited in the NCBI SRA database under
accession number SRP128236 (https://www.ncbi.nlm.nih.gov/sra/SRP128236). The
sequences and annotations of newly discovered loci and isoforms, and the expression level of
genes and isoforms can be downloaded from the Zenodo (https://zenodo.org/) with the
DOI:10.5281/zenodo.2541477.
Author contributions
C.M. and S.X. designed the experiments; X.W., S.C., D.L., Y.C., Y.L., Z.L. and X.N.
performed analyses of the hybrid sequencing data; X.S. carried out the RT-PCR; X.W., C.M.
and S.X. wrote the manuscript. W.S. and Q.S. contributed to experiment design and
manuscript revision.
Short supporting legends
Supplemental Figure 1. Visualization of local and global PID definitions.
Supplemental Figure 2. Schematic of the seven groups of PacBio isoforms.
Supplemental Figure 3. RT-PCR validation of novel gene loci and novel AS isoforms
identified by PacBio.
Supplemental Figure 4. The GO and KEGG annotation of novel isoforms that have BLAST
hits in the NR, GO or KEGG databases.
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Supplemental Figure 5. Comparison of DEGs among the five HS treatment time points in
leaves and grain.
Supplemental Figure 6. Differences in HS response between leaves and grain.
Supplemental Figure 7. Comparison of DSGs among the five HS treatment time points in
leaves and grain.
Supplemental Figure 8. Venn diagram of DEGs (a) and DSGs (b) between leaves and grain at
different time points.
Supplemental Figure 9. Visualization and number of alternative splicing modes.
Supplemental Figure 10. Distribution of the expression levels of the isoforms (FPKM ≥ 1.0)
generated by the four AS modes in the DSGs.
Supplemental Figure 11. The expression variation patterns of each member of each HSF
subfamily.
Supplemental Figure 12. The expression variation patterns of each member of the HSP20,
HSP70, HSP90 and HSP100 families.
Supplemental Figure 13. HS-responsive TFs.
Supplemental Figure 14. The thermal conductivity and signaling duration in leaves.
Supplemental Figure 15. The thermal conductivity and signaling duration in grain.
Supplemental Figure 16. Comparison of DEGs and DSGs at each HS treatment time point in
leaves and grain.
Supplemental Figure 17. Schematic representation of the protein processing in endoplasmic
reticulum pathway exported from the KEGG database.
Supplemental Figure 18. Schematic representation of the spliceosome pathway exported from
the KEGG database.
Supplemental Figure 19. Distribution of DEGs and DSGs among the three wheat subgenomes
at each HS treatment time point.
Supplemental Figure 20. Heatmaps display the DEGs in each homologous triplet at each time
point.
Supplemental Figure 21. Heatmaps display the DSGs in each homologous triplet at each time
point.
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Supplemental Table 1. General properties of the reads produced by Illumina sequencing and
PacBio sequencing.
Supplemental Table 2. The alignment of FLNC reads with the IWGSC RefSeq v1.0.
Supplemental Table 3. The gene and isoform annotations of the IWGSC RefSeq v1.0 and
PacBio data.
Supplemental Table 4. The genes and isoforms identified in the PacBio data.
Supplemental Table 5. The BLAST hits of the newly discovered transcripts among the public
databases.
Supplemental Table 6. The domain annotation and enrichment of newly identified loci in
InterPro database.
Supplemental Table 7. The ratio of repeat sequences in newly discovered gene regions.
Supplemental Table 8. The annotation of the newly discovered loci.
Supplemental Table 9. The list of lncRNAs and their HS responses.
Supplemental Table 10. The list of DEGs.
Supplemental Table 11. The KEGG and GO enrichment analysis of genes which show
different HS response between leaves and grain.
Supplemental Table 12. The list of DSGs.
Supplemental Table 13. The number and ratio of the four splicing modes in all PacBio
isoforms or in the isoforms generated by DSGs.
Supplemental Table 14. The classification of HSFs and their response to HS.
Supplemental Table 15. The classification of HSPs and their response to HS.
Supplemental Table 16. The KEGG and GO enrichment analysis of DEGs.
Supplemental Table 17. The KEGG and GO enrichment analysis of DEG clusters in leaves
and grain.
Supplemental Table 18. The KEGG and GO enrichment analysis of DSGs.
Supplemental Table 19. The KEGG and GO enrichment analysis of DEG- and DSG-specific
genes and overlapping genes between DEGs and DSGs.
Supplemental Table 20. The list of identified homologous triplets.
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Supplemental Table 21. The KEGG and GO enrichment analysis of distinct categories based
on the differential HS responses between the A-, B- and D-homeologues.
References
Abdel-Ghany, S.E., Hamilton, M., Jacobi, J.L., Ngam, P., Devitt, N., Schilkey, F., Ben-Hur, A. and Reddy, A.S. (2016) A survey of the sorghum transcriptome using single-molecule long reads. Nat Commun, 7:11706.
Appels, R., Eversole, K., Feuillet, C., Keller, B., Rogers, J., Stein, N., Pozniak, C.J., Stein, N., Choulet, F., Distelfeld, A., Eversole, K., Poland, J., Rogers, J., Ronen, G., Sharpe, A.G., Pozniak, C., Ronen, G., Stein, N., Barad, O., Baruch, K., Choulet, F., Keeble-Gagnere, G., Mascher, M., Sharpe, A.G., Ben-Zvi, G., Josselin, A.A., Stein, N., Mascher, M., Himmelbach, A., Choulet, F., Keeble-Gagnere, G., Mascher, M., Rogers, J., Balfourier, F., Gutierrez-Gonzalez, J., Hayden, M., Josselin, A.A., Koh, C., Muehlbauer, G., Pasam, R.K., Paux, E., Pozniak, C.J., Rigault, P., Sharpe, A.G., Tibbits, J., Tiwari, V., Choulet, F., Keeble-Gagnere, G., Mascher, M., Josselin, A.A., Rogers, J., Spannagl, M., Choulet, F., Lang, D., Gundlach, H., Haberer, G., Keeble-Gagnere, G., Mayer, K.F.X., Ormanbekova, D., Paux, E., Prade, V., Simkova, H., Wicker, T., Choulet, F., Spannagl, M., Swarbreck, D., Rimbert, H., Felder, M., Guilhot, N., Gundlach, H., Haberer, G., Kaithakottil, G., Keilwagen, J., Lang, D., Leroy, P., Lux, T., Mayer, K.F.X., Twardziok, S., Venturini, L., Appels, R., Rimbert, H., Choulet, F., Juhasz, A., Keeble-Gagnere, G., Choulet, F., Spannagl, M., Lang, D., Abrouk, M., Haberer, G., Keeble-Gagnere, G., Mayer, K.F.X., Wicker, T., Choulet, F., Wicker, T., Gundlach, H., Lang, D., Spannagl, M., Lang, D., Spannagl, M., Appels, R., Fischer, I., Uauy, C., Borrill, P., Ramirez-Gonzalez, R.H., Appels, R., Arnaud, D., Chalabi, S., Chalhoub, B., Choulet, F., Cory, A., Datla, R., Davey, M.W., Hayden, M., Jacobs, J., Lang, D., Robinson, S.J., Spannagl, M., Steuernagel, B., Tibbits, J., Tiwari, V., van Ex, F., Wulff, B.B.H., Pozniak, C.J., Robinson, S.J., Sharpe, A.G., Cory, A., Benhamed, M., Paux, E., Bendahmane, A., Concia, L., Latrasse, D., Rogers, J., Jacobs, J., Alaux, M., Appels, R., Bartos, J., Bellec, A., Berges, H., Dolezel, J., Feuillet, C., Frenkel, Z., Gill, B., Korol, A., Letellier, T., Olsen, O.A., Simkova, H., Singh, K., Valarik, M., van der Vossen, E., Vautrin, S., Weining, S., Korol, A., Frenkel, Z., Fahima, T., Glikson, V., Raats, D., Rogers, J., Tiwari, V., Gill, B., Paux, E., Poland, J., Dolezel, J., Cihalikova, J., Simkova, H., Toegelova, H., Vrana, J., Sourdille, P., Darrier, B., Appels, R., Spannagl, M., Lang, D., Fischer, I., Ormanbekova, D., Prade, V., Barabaschi, D., Cattivelli, L., Hernandez, P., Galvez, S., Budak, H., Steuernagel, B., Jones, J.D.G., Witek, K., Wulff, B.B.H., Yu, G., Small, I., Melonek, J., Zhou, R., Juhasz, A., Belova, T., Appels, R., Olsen, O.A., Kanyuka, K., King, R., Nilsen, K., Walkowiak, S., Pozniak, C.J., Cuthbert, R., Datla, R., Knox, R., Wiebe, K., Xiang, D., Rohde, A., Golds, T., Dolezel, J., Cizkova, J., Tibbits, J., Budak, H., Akpinar, B.A., Biyiklioglu, S., Muehlbauer, G., Poland, J., Gao, L., Gutierrez-Gonzalez, J., N'Daiye, A., Dolezel, J., Simkova, H., Cihalikova, J., Kubalakova, M., Safar, J., Vrana, J., Berges, H., Bellec, A., Vautrin, S., Alaux, M., Alfama, F., Adam-Blondon, A.F., Flores, R., Guerche, C., Letellier, T., Loaec, M., Quesneville, H., Pozniak, C.J., Sharpe, A.G., Walkowiak, S., Budak, H., Condie, J., Ens, J., Koh, C., Maclachlan, R., Tan, Y., Wicker, T., Choulet, F., Paux, E., Alberti, A., Aury, J.M., Balfourier, F., Barbe, V., Couloux, A., Cruaud, C., Labadie, K., Mangenot, S., Wincker, P., Gill, B., Kaur, G., Luo, M., Sehgal, S., Singh, K., Chhuneja, P., Gupta, O.P., Jindal, S., Kaur, P., Malik, P., Sharma, P., Yadav, B., Singh, N.K., Khurana, J., Chaudhary, C., Khurana, P., Kumar, V., Mahato, A., Mathur, S., Sevanthi, A., Sharma, N., Tomar, R.S., Rogers, J., Jacobs, J., Alaux, M., Bellec, A., Berges, H., Dolezel, J., Feuillet, C., Frenkel, Z., Gill, B., Korol, A., van der Vossen, E., Vautrin, S., Gill, B., Kaur, G., Luo, M., Sehgal, S., Bartos, J., Holusova, K., Plihal, O., Clark, M.D., Heavens, D., Kettleborough, G., Wright, J., Valarik, M., Abrouk, M., Balcarkova, B., Holusova, K., Hu, Y., Luo, M., Salina, E., Ravin, N., Skryabin, K., Beletsky, A., Kadnikov, V., Mardanov, A., Nesterov, M., Rakitin, A., Sergeeva, E., Handa, H., Kanamori, H., Katagiri, S., Kobayashi, F., Nasuda, S., Tanaka, T., Wu, J., Appels, R., Hayden, M., Keeble-Gagnere, G., Rigault, P., Tibbits, J., Olsen, O.A., Belova, T., Cattonaro, F., Jiumeng, M., Kugler, K., Mayer, K.F.X., Pfeifer, M., Sandve, S., Xun, X., Zhan, B., Simkova, H., Abrouk, M., Batley, J., Bayer, P.E., Edwards, D., Hayashi, S., Toegelova, H., Tulpova, Z., Visendi, P., Weining, S., Cui, L., Du, X., Feng, K., Nie, X., Tong, W., Wang, L., Borrill, P., Gundlach, H., Galvez, S., Kaithakottil, G., Lang, D., Lux, T., Mascher, M., Ormanbekova, D., Prade, V., Ramirez-Gonzalez, R.H., Spannagl, M.,
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Stein, N., Uauy, C., Venturini, L., Stein, N., Appels, R., Eversole, K., Rogers, J., Borrill, P., Cattivelli, L., Choulet, F., Hernandez, P., Kanyuka, K., Lang, D., Mascher, M., Nilsen, K., Paux, E., Pozniak, C.J., Ramirez-Gonzalez, R.H., Simkova, H., Small, I., Spannagl, M., Swarbreck, D. and Uauy, C. (2018) Shifting the limits in wheat research and breeding using a fully annotated reference genome. Science, 361.
Benjamini, Y. and Hochberg, Y. (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the royal statistical society, 57, 289-300.
Bokszczanin, K.L., Network, S.P.T.I.T. and Consortium, S.F. (2013) Perspectives on deciphering mechanisms underlying plant heat stress response and thermotolerance. Frontiers in plant science, 4: 315.
Bork, P., Doerks, T., Springer, T.A. and Snel, B. (1999) Domains in plexins: links to integrins and transcription factors. Trends in biochemical sciences, 24, 261-263.
Campbell, J.L., Klueva, N.Y., Zheng, H.-g., Nieto-Sotelo, J., Ho, T.-H. and Nguyen, H.T. (2001) Cloning of new members of heat shock protein HSP101 gene family in wheat (Triticum aestivum (L.) Moench) inducible by heat, dehydration, and ABA. Biochim Biophys Acta, 1517, 270-277.
Carrasco, J.L., Ancillo, G., Castello, M.J. and Vera, P. (2005) A novel DNA-binding motif, hallmark of a new family of plant transcription factors. Plant Physiol, 137, 602-606.
Chang, C.Y., Lin, W.D. and Tu, S.L. (2014) Genome-Wide Analysis of Heat-Sensitive Alternative Splicing in Physcomitrella patens. Plant Physiol, 165, 826-840.
Conesa, A., Gotz, S., Garcia-Gomez, J.M., Terol, J., Talon, M. and Robles, M. (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 21, 3674-3676.
Ding, F., Cui, P., Wang, Z., Zhang, S., Ali, S. and Xiong, L. (2014) Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis. BMC Genomics, 15, 431.
Du, L. and Poovaiah, B.W. (2004) A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. Plant Mol Biol, 54, 549-569.
Dubcovsky, J. and Dvorak, J. (2007) Genome plasticity a key factor in the success of polyploid wheat under domestication. Science, 316, 1862-1866.
Farooq, M., Bramley, H., Palta, J.A. and Siddique, K.H. (2011) Heat stress in wheat during reproductive and grain-filling phases. Critical Reviews in Plant Sciences, 30, 491-507.
Feldman, M. and Levy, A.A. (2009) Genome evolution in allopolyploid wheat--a revolutionary reprogramming followed by gradual changes. J Genet Genomics, 36, 511-518.
Florea, L., Song, L. and Salzberg, S.L. (2013) Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues. F1000Research, 2, 188.
Goodwin, S., McPherson, J.D. and McCombie, W.R. (2016) Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet, 17, 333-351.
Guo, W., Calixto, C.P.G., Brown, J.W.S. and Zhang, R. (2017) TSIS: an R package to infer alternative splicing isoform switches for time-series data. Bioinformatics, 33, 3308-3310.
Hackl, T., Hedrich, R., Schultz, J. and Förster, F. (2014) proovread: large-scale high-accuracy PacBio correction through iterative short read consensus. Bioinformatics, 30, 3004-3011.
Hennig, L. (2012) Plant gene regulation in response to abiotic stress. Biochim Biophys Acta, 1819, 85. Kanei-Ishii, C., Tanikawa, J., Nakai, A., Morimoto, R.I. and Ishii, S. (1997) Activation of heat shock
transcription factor 3 by c-Myb in the absence of cellular stress. Science, 277, 246-248. Kielbowicz-Matuk, A. (2012) Involvement of plant C2H2-type zinc finger transcription factors in stress
responses. Plant Science, 185, 78-85. Kotak, S., Larkindale, J., Lee, U., von Koskull-Döring, P., Vierling, E. and Scharf, K.-D. (2007) Complexity of the
heat stress response in plants. Curr Opin Plant biol, 10, 310-316. Lesk, C., Rowhani, P. and Ramankutty, N. (2016) Influence of extreme weather disasters on global crop
production. Nature, 529, 84-87. Li, B. and Dewey, C.N. (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a
reference genome. BMC Bioinformatics, 12, 323. Li, W., Lin, W.D., Ray, P., Lan, P. and Schmidt, W. (2013) Genome-wide detection of condition-sensitive
alternative splicing in Arabidopsis roots. Plant Physiol, 162, 1750-1763.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Ling, Z., Zhou, W., Baldwin, I.T. and Xu, S. (2015) Insect herbivory elicits genome-wide alternative splicing responses in Nicotiana attenuata. The Plant journal, 84, 228-243.
Liu, J., Sun, N., Liu, M., Liu, J., Du, B., Wang, X. and Qi, X. (2013) An autoregulatory loop controlling Arabidopsis HsfA2 expression: role of heat shock-induced alternative splicing. Plant Physiol, 162, 512-521.
Liu, Q., Wang, Z., Xu, X., Zhang, H. and Li, C. (2015a) Genome-wide analysis of C2H2 Zinc-finger family transcription factors and their responses to abiotic stresses in poplar (Populus trichocarpa). PloS one, 10, e0134753.
Liu, Z., Qin, J., Tian, X., Xu, S., Wang, Y., Li, H., Wang, X., Peng, H., Yao, Y., Hu, Z., Ni, Z., Xin, M. and Sun, Q. (2018) Global Profiling of Alternative Splicing Landscape Responsive to Drought, Heat and Their Combination in Wheat (Triticum asetivum L.). Plant Biotechnol J, 16: 714-726.
Liu, Z., Xin, M., Qin, J., Peng, H., Ni, Z., Yao, Y. and Sun, Q. (2015b) Temporal transcriptome profiling reveals expression partitioning of homeologous genes contributing to heat and drought acclimation in wheat (Triticum aestivum L.). BMC Plant Biol, 15, 152.
Lobell, D.B. and Tebaldi, C. (2014) Getting caught with our plants down: the risks of a global crop yield slowdown from climate trends in the next two decades. Environ Res Lett, 9, 074003.
Mangelsen, E., Kilian, J., Harter, K., Jansson, C., Wanke, D. and Sundberg, E. (2011) Transcriptome analysis of high-temperature stress in developing barley caryopses: early stress responses and effects on storage compound biosynthesis. Mol Plant, 4, 97-115.
Mitsuda, N., Hisabori, T., Takeyasu, K. and Sato, M.H. (2004) VOZ; isolation and characterization of novel vascular plant transcription factors with a one-zinc finger from Arabidopsis thaliana. Plant & Cell Physiol, 45, 845-854.
Nollen, E.A. and Morimoto, R.I. (2002) Chaperoning signaling pathways: molecular chaperones as stress-sensingheat shock'proteins. J Cell Sci, 115, 2809-2816.
Nussbaumer, T., Warth, B., Sharma, S., Ametz, C., Bueschl, C., Parich, A., Pfeifer, M., Siegwart, G., Steiner, B., Lemmens, M., Schuhmacher, R., Buerstmayr, H., Mayer, K.F., Kugler, K.G. and Schweiger, W. (2015) Joint Transcriptomic and Metabolomic Analyses Reveal Changes in the Primary Metabolism and Imbalances in the Subgenome Orchestration in the Bread Wheat Molecular Response to Fusarium graminearum. G3, 5, 2579-2592.
Pfeifer, M., Kugler, K.G., Sandve, S.R., Zhan, B., Rudi, H., Hvidsten, T.R., Mayer, K.F. and Olsen, O.A. (2014) Genome interplay in the grain transcriptome of hexaploid bread wheat. Science, 345, 1250091.
Powell, J.J., Fitzgerald, T.L., Stiller, J., Berkman, P.J., Gardiner, D.M., Manners, J.M., Henry, R.J. and Kazan, K. (2017) The defence-associated transcriptome of hexaploid wheat displays homoeolog expression and induction bias. Plant Biotechnol J, 15, 533-543.
Rhoads, A. and Au, K.F. (2015) PacBio sequencing and its applications. Genomics, proteomics & Bioinformatics, 13, 278-289.
Robinson, M.D., McCarthy, D.J. and Smyth, G.K. (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26, 139-140.
Saidi, Y., Finka, A. and Goloubinoff, P. (2011) Heat perception and signalling in plants: a tortuous path to thermotolerance. New Phytol, 190, 556-565.
Scharf, K.-D., Berberich, T., Ebersberger, I. and Nover, L. (2012) The plant heat stress transcription factor (Hsf) family: structure, function and evolution. Biochim Biophys Acta, 1819, 104-119.
Shen, Y., Zhou, Z., Wang, Z., Li, W., Fang, C., Wu, M., Ma, Y., Liu, T., Kong, L.-A., Peng, D.-L. and Tian, Z. (2014) Global Dissection of Alternative Splicing in Paleopolyploid Soybean. The Plant Cell, 26, 996-1008.
Soneson, C., Love, M.I. and Robinson, M.D. (2015) Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research, 4.
Sugio, A., Dreos, R., Aparicio, F. and Maule, A.J. (2009) The cytosolic protein response as a subcomponent of the wider heat shock response in Arabidopsis. The Plant Cell, 21, 642-654.
Tack, J., Barkley, A. and Nalley, L.L. (2015) Effect of warming temperatures on US wheat yields. Proc Natl Acad Sci U S A, 112, 6931-6936.
Tingley, M.P. and Huybers, P. (2013) Recent temperature extremes at high northern latitudes unprecedented in the past 600 years. Nature, 496, 201-205.
Trapnell, C., Pachter, L. and Salzberg, S.L. (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics, 25, 1105-1111.
Wahid, A., Gelani, S., Ashraf, M. and Foolad, M.R. (2007) Heat tolerance in plants: an overview. Environmental and Experimental botany, 61, 199-223.
Acc
epte
d A
rtic
le
This article is protected by copyright. All rights reserved.
Wang, B., Tseng, E., Regulski, M., Clark, T.A., Hon, T., Jiao, Y., Lu, Z., Olson, A., Stein, J.C. and Ware, D. (2016) Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing. Nat Commun, 7, 11708.
Wang, L.-J. and Li, S.-H. (2006) Salicylic acid-induced heat or cold tolerance in relation to Ca 2+ homeostasis and antioxidant systems in young grape plants. Plant Science, 170, 685-694.
Wang, L., Park, H.J., Dasari, S., Wang, S., Kocher, J.-P. and Li, W. (2013) CPAT: Coding-Potential Assessment Tool using an alignment-free logistic regression model. Nucleic Acids Res, 41, e74-e74.
Wang, M., Wang, P., Liang, F., Ye, Z., Li, J., Shen, C., Pei, L., Wang, F., Hu, J., Tu, L., Lindsey, K., He, D. and Zhang, X. (2017a) A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation. New Phytol, 217, 163-178.
Wang, X., Hou, L., Lu, Y., Wu, B., Gong, X., Liu, M., Wang, J., Sun, Q., Vierling, E. and Xu, S. (2018a) Metabolic adaptation of wheat grain contributes to stable filling rate under heat stress. Journal of Experimental Botany, ery303-ery303.
Wang, X., Shi, X., Chen, S., Ma, C. and Xu, S. (2018b) Evolutionary origin, gradual accumulation and functional divergence of heat shock factor gene family with plant evolution. Frontiers in Plant Science, 9, 71.
Wang, X., Wang, R., Ma, C., Shi, X., Liu, Z., Wang, Z., Sun, Q., Cao, J. and Xu, S. (2017b) Massive expansion and differential evolution of small heat shock proteins with wheat (Triticum aestivum L.) polyploidization. Scientific Reports, 7, 2158.
Wilkins, O., Hafemeister, C., Plessis, A., Holloway-Phillips, M.-M., Pham, G.M., Nicotra, A.B., Gregorio, G.B., Jagadish, S.V.K., Septiningsih, E.M., Bonneau, R. and Purugganan, M. (2016) EGRINs (Environmental Gene Regulatory Influence Networks) in Rice That Function in the Response to Water Deficit, High Temperature, and Agricultural Environments. The Plant Cell, 28, 2365-2384.
Wollenweber, B., Porter, J.R. and Schellberg, J. (2003) Lack of Interaction between Extreme High-Temperature Events at Vegetative and Reproductive Growth Stages in Wheat. Journal of Agronomy and Crop Science, 189, 142-150.
Wong, J.J., Ritchie, W., Ebner, O.A., Selbach, M., Wong, J.W., Huang, Y., Gao, D., Pinello, N., Gonzalez, M., Baidya, K., Thoeng, A., Khoo, T.L., Bailey, C.G., Holst, J. and Rasko, J.E. (2013) Orchestrated intron retention regulates normal granulocyte differentiation. Cell, 154, 583-595.
Wu, T.D. and Watanabe, C.K. (2005) GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics, 21, 1859-1875.
Xie, C., Mao, X., Huang, J., Ding, Y., Wu, J., Dong, S., Kong, L., Gao, G., Li, C.Y. and Wei, L. (2011) KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res, 39, W316-322.
Zhao, C., Liu, B., Piao, S., Wang, X., Lobell, D.B., Huang, Y., Huang, M., Yao, Y., Bassu, S. and Ciais, P. (2017) Temperature increase reduces global yields of major crops in four independent estimates. Proc Natl Acad Sci U S A, 114, 9326-9331.
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Figure legends
Figure 1. Experimental workflow. (a) Wheat plants (T. aestivum cv. Chinese Spring) at 15
days after anthesis were subjected to HS (37C). The flag leaves and filling grain were
sampled at 0 m, 5 m, 10 m, 30 m, 1 h and 4 h under HS. A total of 36 samples (six time
points for each of the two organs, three biological replicates per time point) were sequenced
using second-generation sequencing, and two mixed samples (the RNAs of 18 samples from
each organ mixed in equal volume) were sequenced using third-generation sequencing. (b)
Bioinformatics pipeline for analyzing the hybrid sequencing data. Sequencing errors in FLNC
reads were corrected with short reads. FLNC reads before and after error correction were
mapped in parallel to IWGSC RefSeq v1.0, and the read with the best genomic match (see
Methods) was retained in the downstream analysis. (c) Comparison of the transcript length
between the IWGSC RefSeq v1.0 annotation and the PacBio data. (d) Comparison of the
isoform number between the IWGSC RefSeq v1.0 annotation and the PacBio data. (e)
Structure comparison of the IWGSC RefSeq v1.0 and PacBio transcripts.
Figure 2. CIRCOS visualization of different data at the genome-wide level. The density was
calculated in a 10-Mb sliding window. (a) Karyotype of the wheat genome. (b) Comparison
of transcript density between the IWGSC RefSeq v1.0 annotation and the PacBio data. From
the upper to lower tracks: transcripts in IWGSC RefSeq v1.0, transcripts in grain and
transcripts in leaves. (c–g) Distribution of HS-responsive genes following HS treatment for 4
h, 1 h, 30 m, 10 m and 5 m. From the upper to lower tracks in each part: the HS-responsive
genes in grain with transcriptional regulation, the HS-responsive genes in leaves with
transcriptional regulation, the HS-responsive genes in grain with AS regulation and the HS-
responsive genes in leaves with AS regulation. (h,i) Distribution of LncRNAs in grain (h) and
leaves (i). (j) Linkage of fusion transcripts: intra-chromosome (green), inter-chromosome in
the same subgenome (red) and inter-chromosome in different subgenomes (blue).
Figure 3. Identification of DEGs and DSGs at each time point in leaves and grain. Number of
DEGs (a) and DSGs (c). The x-axis represents the HS treatment time points and the y-axis
represents the HS-responsive gene number. Light blue and light red represent the number of
DEGs and DSGs that were newly discovered loci from the PacBio data, respectively. (b)
Number of different HS response genes between leaves and grain. Light blue represents
newly discovered loci from the PacBio data. (d) Venn diagram of DSGs in leaves and grain.
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Figure 4. Rapid changes of DEGs and DSGs in response to HS. (a,b) Histograms plots of the
time points at which the DEGs (a) and DSGs (b) first showed a significant difference in
leaves and grain. Each gene is represented only once in each histogram. (c) Frequency over
time of isoform switches (where the relative abundance of different isoforms is reversed in
response to HS) in the time-course transcriptomes. (d,e) Expression profiles of pre-mRNA-
splicing factor SF2 (d) and HSFA6 (e). The isoform switch events were marked with black
circles. For clarity, only the transcripts that were involved in isoform switches were plotted.
The isoforms whose names start with “chr” were novel isoforms identified from the PacBio
data.
Figure 5. Differences in timing of diverse TFs in the heat signaling and early heat response.
(a,b) Heatmaps showing the fold enrichment of TFs in response to HS with transcriptional
regulation (a) and AS regulation (b). Only significantly enriched families (q < 0.05) are
indicated. The x-axis represents HS-treated samples and the y-axis represents the TF families.
“L” and “G” in the sample names represent leaf and grain, respectively.
Figure 6. Heat signaling and early heat response processes. (a,b) Heatmaps showing the fold
enrichment of enriched KEGG pathways for DEGs (a) and DSGs (b). Only significantly
enriched pathways (q < 0.05) are indicated. The full list of enriched pathways is presented in
Tables S16 and S18. The x-axis represents HS treatment time points and the y-axis represents
enriched KEGG pathways. “L” and “G” in the sample names represent leaf and grain,
respectively.
Figure 7. Overlap of DEGs and DSGs, and a HSF coding gene that responds to HS with both
transcriptional regulation and AS regulation. (a) Overlap of DEGs and DSGs in leaves. (b)
Overlap of DEGs and DSGs in grain. (c) Schematic representation of the isoforms produced
by TraesCS5D01G393200 (a member of the HSFA2 subfamily). Exons are represented as
blue boxes and introns as lines. The isoforms whose names start with “Chr” were novel
isoforms identified from the PacBio data. The red triangle indicates the location of an in-
frame premature termination codon in the intron. For the right part, the numbers in rectangles
represent average FPKM values of three replicates at each time point in leaves. The heatmap
shows the fold change of each isoform at different time points (0 m time point was used as a
control).
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Figure 8. Subgenome bias in the HS response. (a,b) Heatmaps showing the fold change in the
expression of each gene in each homologous triplet at the 30 m time point in leaves (a) and at
the 1 h time point in grain (b). Only the triplets that contain DEGs are displayed. For the
symbols on the x-axis, “A”, “B” and “D” represent the A-, B- and D-homeologues in the
triplets, respectively. “L” and “G” represent the leaves and grain, respectively. In the right
part, the heatmaps display the pairwise ratio of the fold changes between the A-, B- and D-
homeologues. The green, purple and orange represent the different responses of the A-, B-
and D-homeologues in each pairwise comparison. A vs. B: the comparison between the A-
and B-homeologues, A vs. D: the comparison between the A- and D-homeologues, and B vs.
D: the comparison between the B- and D-homeologues. (c,d) Heatmaps display the DSGs in
each homologous triplet at the 30 m time point in leaves (c) and at the 1 h time point in grain
(d). Only the triplets that contain DSGs are displayed. The symbol “1” indicates that the gene
is a DSG and the symbol “0” indicates that the gene is not a DSG.
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This article is protected by copyright. All rights reserved.
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This article is protected by copyright. All rights reserved.
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