Heat stress (HS) is a major abiotic stress that negatively impacts crop yields across the globe. Plants respond to elevated temperatures by changing gene expression, mediated by transcription factors (TFs) functioning to enhance HS tolerance. The involvement of Group I bZIP TFs in the heat stress response (HSR) is not known. In this study, bZIP18 and bZIP52 were investigated for their possible role in the HSR. Localization experiments revealed their nuclear accumulation following heat stress, which was found to be triggered by dephosphorylation. Both TFs were found to possess two motifs containing serine residues that are candidates for phosphorylation. These motifs are recognized by 14-3-3 proteins, and bZIP18 and bZIP52 were found to bind 14-3-3 ε, the interaction of which sequesters them to the cytoplasm. Mutation of both residues abolished 14-3-3 ε interaction and led to a strict nuclear localization for both TFs. RNA-seq analysis revealed coordinated downregulation of several metabolic pathways including energy metabolism and translation, and upregulation of numerous lncRNAs in particular. These results support the idea that bZIP18 and bZIP52 are sequestered to the cytoplasm under control conditions, and that heat stress leads to their re-localization to nuclei, where they jointly regulate gene expression.

Department of Experimental Plant Biology Faculty of Science Charles University Viničná 5 128 44 Prague 2 Czech Republic

Laboratory of Pollen Biology Institute of Experimental Botany of the Czech Academy of Sciences Rozvojová 263 165 02 Prague 6 Czech Republic

Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology Masaryk University Kamenice 753 5 62 500 Brno Czech Republic

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Hassan M.U., Chattha M.U., Khan I., Chattha M.B., Barbanti L., Aamer M., Iqbal M.M., Nawaz M., Mahmood A., Ali A., et al. Heat stress in cultivated plants: Nature, impact, mechanisms, and mitigation strategies—A review. Plant Biosyst. 2020 doi: 10.1080/11263504.2020.1727987. DOI

Priya M., Sharma L., Kaur R., Bindumadhava H., Nair R.M., Siddique K.H.M., Nayyar H. GABA (γ-aminobutyric acid), as a thermo-protectant, to improve the reproductive function of heat-stressed mungbean plants. Sci. Rep. 2019;9 doi: 10.1038/s41598-019-44163-w. PubMed DOI PMC

Pareek A., Dhankher O.P., Foyer C.H. Mitigating the impact of climate change on plant productivity and ecosystem sustainability. J. Exp. Bot. 2020;71:451–456. doi: 10.1093/jxb/erz518. PubMed DOI PMC

IPCC . Summary for Policymakers. In: Stocker T.F., Qin D., Plattner G.-K., Tignor M., Allen S.K., Boschung J., Nauels A., Xia Y., Bex V., Midgley P.M., editors. Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press; Cambridge, UK: New York, NY, USA: 2013.

Hasanuzzaman M., Nahar K., Alam M.M., Roychowdhury R., Fujita M. Physiological, Biochemical, and Molecular Mechanisms of Heat Stress Tolerance in Plants. Int. J. Mol. Sci. 2013;14:9643–9684. doi: 10.3390/ijms14059643. PubMed DOI PMC

Mittler R., Finka A., Goloubinoff P. How do plants feel the heat? Trends Biochem. Sci. 2012;37 doi: 10.1016/j.tibs.2011.11.007. PubMed DOI

Baena-González E., Rolland F., Thevelein J.M., Sheen J. A central integrator of transcription networks in plant stress and energy signalling. Nature. 2007;448:938–942. doi: 10.1038/nature06069. PubMed DOI

Iwata Y., Fedoroff N.V., Koizumi N. Arabidopsis bZIP60 Is a Proteolysis-Activated Transcription Factor Involved in the Endoplasmic Reticulum Stress Response. Plant Cell. 2008;20:3107–3121. doi: 10.1105/tpc.108.061002. PubMed DOI PMC

Smykowski A., Zimmermann P., Zentgraf U. G-Box Binding Factor1 Reduces CATALASE2 Expression and Regulates the Onset of Leaf Senescence in Arabidopsis. Plant Physiol. 2010;153:1321–1331. doi: 10.1104/pp.110.157180. PubMed DOI PMC

Abe M., Kobayashi Y., Yamamoto S., Daimon Y., Yamaguchi A., Ikeda Y., Ichinoki H., Notaguchi M., Goto K., Araki T. FD, a bZIP Protein Mediating Signals from the Floral Pathway Integrator FT at the Shoot Apex. Science. 2005;309:1052–1056. doi: 10.1126/science.1115983. PubMed DOI

Gibalová A., Renák D., Matczuk K., Dupl’áková N., Cháb D., Twell D., Honys D. AtbZIP34 is required for Arabidopsis pollen wall patterning and the control of several metabolic pathways in developing pollen. Plant Mol. Biol. 2009;70:581–601. doi: 10.1007/s11103-009-9493-y. PubMed DOI

Iven T., Strathmann A., Böttner S., Zwafink T., Heinekamp T., Guivarc’h A., Roitsch T., Dröge-Laser W. Homo- and heterodimers of tobacco bZIP proteins counteract as positive or negative regulators of transcription during pollen development-Iven-2010-The Plant Journal-Wiley Online Library. Plant J. 2010;63:155–166. doi: 10.1111/j.1365-313X.2010.04230.x. PubMed DOI

Gibalová A., Steinbachová L., Hafidh S., Bláhová V., Gadiou Z., Michailidis C., Műller K., Pleskot R., Dupľáková N., Honys D. Characterization of pollen-expressed bZIP protein interactions and the role of ATbZIP18 in the male gametophyte. Plant Reprod. 2017;30:1–17. doi: 10.1007/s00497-016-0295-5. PubMed DOI

Alonso R., Oñate-Sánchez L., Weltmeier F., Ehlert A., Diaz I., Dietrich K., Vicente-Carbajosa J., Dröge-Laser W. A pivotal role of the basic leucine zipper transcription factor bZIP53 in the regulation of Arabidopsis seed maturation gene expression based on heterodimerization and protein complex formation. Plant Cell. 2009;21:1747–1761. doi: 10.1105/tpc.108.062968. PubMed DOI PMC

Fujita Y., Fujita M., Satoh R., Maruyama K., Parvez M.M., Seki M., Hiratsu K., Ohme-Takagi M., Shinozaki K., Yamaguchi-Shinozaki K. AREB1 Is a Transcription Activator of Novel ABRE-Dependent ABA Signaling That Enhances Drought Stress Tolerance in Arabidopsis. Plant Cell. 2005;17:3470–3488. doi: 10.1105/tpc.105.035659. PubMed DOI PMC

Dröge-Laser W., Snoek B.L., Snel B., Weiste C. The Arabidopsis bZIP transcription factor family-an update. Curr. Opin. Plant Biol. 2018;45:36–49. doi: 10.1016/j.pbi.2018.05.001. PubMed DOI

Bao Y., Howell S.H. The Unfolded Protein Response Supports Plant Development and Defense as well as Responses to Abiotic Stress. Front. Plant Sci. 2017 doi: 10.3389/fpls.2017.00344. PubMed DOI PMC

Deng Y., Humbert S., Liu J.-X., Srivastava R., Rothstein S.J., Howell S.H. Heat induces the splicing by IRE1 of a mRNA encoding a transcription factor involved in the unfolded protein response in Arabidopsis. Proc. Natl. Acad. Sci. USA. 2011;107:7247–7252. doi: 10.1073/pnas.1102117108. PubMed DOI PMC

Nagashima Y., Mishiba K.-I., Suzuki E., Shimada Y., Iwata Y., Koizumi N. Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor. Sci. Rep. 2011;1:1–10. doi: 10.1038/srep00029. PubMed DOI PMC

Herath V., Gayral M., Adhikari N., Miller R., Verchot J. Genome-wide identification and characterization of Solanum tuberosum BiP genes reveal the role of the promoter architecture in BiP gene diversity. Sci. Rep. 2020;10:1–14. doi: 10.1038/s41598-020-68407-2. PubMed DOI PMC

Liu J.-X., Howell S.H. Endoplasmic Reticulum Protein Quality Control and Its Relationship to Environmental Stress Responses in Plants. Plant Cell. 2010;22:2930–2942. doi: 10.1105/tpc.110.078154. PubMed DOI PMC

Liu J.X., Srivastava R., Che P., Howell S.H. Salt stress responses in Arabidopsis utilize a signal transduction pathway related to endoplasmic reticulum stress signaling-Liu-2007-The Plant Journal-Wiley Online Library. Plant J. 2007;51:897–909. doi: 10.1111/j.1365-313X.2007.03195.x. PubMed DOI PMC

Hartmann L., Pedrotti L., Weiste C., Fekete A., Schierstaedt J., Göttler J., Kempa S., Krischke M., Dietrich K., Mueller M.J., et al. Crosstalk between Two bZIP Signaling Pathways Orchestrates Salt-Induced Metabolic Reprogramming in Arabidopsis Roots. Plant Cell. 2015;27 doi: 10.1105/tpc.15.00163. PubMed DOI PMC

Banerjee A., Roychoudhury A. Epigenetic regulation during salinity and drought stress in plants: Histone modifications and DNA methylation. Plant Gene. 2017;11:199–204. doi: 10.1016/j.plgene.2017.05.011. DOI

Sirichandra C., Davanture M., Turk B.E., Zivy M., Valot B., Leung J., Merlot S. The Arabidopsis ABA-Activated Kinase OST1 Phosphorylates the bZIP Transcription Factor ABF3 and Creates a 14-3-3 Binding Site Involved in Its Turnover. PLoS ONE. 2010;5 doi: 10.1371/journal.pone.0013935. PubMed DOI PMC

Tsugama D., Liu S., Takano T. Analysis of Functions of VIP1 and Its Close Homologs in Osmosensory Responses of Arabidopsis thaliana. PLoS ONE. 2014;9 doi: 10.1371/journal.pone.0103930. PubMed DOI PMC

Takeo K., Ito T. Subcellular localization of VIP1 is regulated by phosphorylation and 14-3-3 proteins. FEBS Lett. 2017;591 doi: 10.1002/1873-3468.12686. PubMed DOI

Tsugama D., Yoon H.S., Fujino K., Liu S., Takano T. Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress. J. Exp. Bot. 2019;70:6101–6112. doi: 10.1093/jxb/erz384. PubMed DOI PMC

Djamei A., Pitzschke A., Nakagami H., Rajh I., Hirt H. Trojan Horse Strategy in Agrobacterium Transformation: Abusing MAPK Defense Signaling. Science. 2007;318:453–456. doi: 10.1126/science.1148110. PubMed DOI

Pitzschke A., Djamei A., Teige M., Hirt H. VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression. PNAS. 2009;106:18414–18419. doi: 10.1073/pnas.0905599106. PubMed DOI PMC

Tsugama D., Liu S., Fujino K., Takano T. Possible inhibition of Arabidopsis VIP1-mediated mechanosensory signaling by streptomycin. Plant Signal. Behav. 2018;13 doi: 10.1080/15592324.2018.1521236. PubMed DOI PMC

Kim J.-S., Yamaguchi-Shinozaki K., Shinozaki K. ER-Anchored Transcription Factors bZIP17 and bZIP28 Regulate Root Elongation. Plant Physiol. 2018;176:2221–2230. doi: 10.1104/pp.17.01414. PubMed DOI PMC

Franco-Zorrilla J.M., López-Vidriero I., Carrasco J.L., Godoy M., Vera P., Solano R. DNA-binding specificities of plant transcription factors and their potential to define target genes. Proc. Natl. Acad. Sci. USA. 2014;111:2367–2372. doi: 10.1073/pnas.1316278111. PubMed DOI PMC

O’Malley R.C., Huang S.-s.C., Song L., Lewsey M.G., Bartlett A., Nery J.R., Galli M., Gallavotti A., Ecker J.R. Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape: Cell. Cell. 2016;165:1280–1292. doi: 10.1016/j.cell.2016.04.038. PubMed DOI PMC

Johnson C., Crowther S., Stafford M.J., Campbell D.G., Toth R., MacKintosh C. Bioinformatic and experimental survey of 14-3-3-binding sites. Biochem. J. 2010;427:69–78. doi: 10.1042/BJ20091834. PubMed DOI PMC

Niemiro A., Cysewski2 D., Brzywczy J., Wawrzyńska A., Sieńko M., Poznański J., Sirko A. Similar but Not Identical—Binding Properties of LSU (Response to Low Sulfur) Proteins from Arabidopsis thaliana. Plant Sci. 2020;11 doi: 10.3389/fpls.2020.01246. PubMed DOI PMC

Xing S., Wallmeroth N., Berendzen K.W., Grefen C. Techniques for the Analysis of Protein-Protein Interactions in Vivo. Plant Physiol. 2016;171:727–758. doi: 10.1104/pp.16.00470. PubMed DOI PMC

Leene J.V., Blomme J., Kulkarni S.R., Cannoot B., Winne N.D., Eeckhout D., Persiau G., Slijke E.V.D., Vercruysse L., Bossche R.V., et al. Functional characterization of the Arabidopsis transcription factor bZIP29 reveals its role in leaf and root development. J. Exp. Bot. 2016;67 doi: 10.1093/jxb/erw347. PubMed DOI PMC

Xin M., Wang Y., Yao Y., Song N., Hu Z., Qin D., Xie C., Peng H., Ni Z., Sun Q. Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing. BMC Plant Biol. 2011;11:61. doi: 10.1186/1471-2229-11-61. PubMed DOI PMC

Wang A., Hu J., Gao C., Chen G., Wang B., Lin C., Song L., Ding Y., Zhou G. Genome-wide analysis of long non-coding RNAs unveils the regulatory roles in the heat tolerance of Chinese cabbage (Brassica rapa ssp. chinensis) Sci. Rep. 2019;9:5002. doi: 10.1038/s41598-019-41428-2. PubMed DOI PMC

He X., Guo S., Wang Y., Wang L., Shu S., Sun J. Systematic identification and analysis of heat-stress-responsive lncRNAs, circRNAs and miRNAs with associated co-expression and ceRNA networks in cucumber (Cucumis sativus L.) Physiol. Plant. 2020;168:736–754. doi: 10.1111/ppl.12997. PubMed DOI

Bhatia G., Singh A., Verma D., Sharma S., Singh K. Genome-wide investigation of regulatory roles of lncRNAs in response to heat and drought stress in Brassica juncea (Indian mustard) Environ. Exp. Bot. 2020;171 doi: 10.1016/j.envexpbot.2019.103922. DOI

Jannesar M., Seyedi S.M., Moazzam Jazi M., Niknam V., Ebrahimzadeh H., Botanga C. A genome-wide identification, characterization and functional analysis of salt-related long non-coding RNAs in non-model plant Pistacia vera L. using transcriptome high throughput sequencing. Sci. Rep. 2020;10:5585. doi: 10.1038/s41598-020-62108-6. PubMed DOI PMC

Yan Q., Wu F., Yan Z., Li J., Ma T., Zhang Y., Zhao Y., Wang Y., Zhang J. Differential co-expression networks of long non-coding RNAs and mRNAs in Cleistogenes songorica under water stress and during recovery. BMC Plant Biol. 2019;19:23. doi: 10.1186/s12870-018-1626-5. PubMed DOI PMC

Karimi M., Inzé D., Depicker A. GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 2002;7:193–195. doi: 10.1016/S1360-1385(02)02251-3. PubMed DOI

Nakagawa T., Kurose T., Hino T., Tanaka K., Kawamukai M., Niwa Y., Toyooka K., Matsuoka K., Jinbo T., Kimuraf T. Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J. Biosci. Bioeng. 2007;104:34–41. doi: 10.1263/jbb.104.34. PubMed DOI

Grefen C., Blatt M.R. A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC) BioTechniques. 2018;53 doi: 10.2144/000113941. PubMed DOI

Wang Z.-P., Xing H.-L., Dong L., Zhang H.-Y., Han C.-Y., Wang X.-C., Chen Q.-J. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 2015;16:1–12. doi: 10.1186/s13059-015-0715-0. PubMed DOI PMC

Gehl C., Waadt R., Kudla J., Mendel R.-R., Hänsch R. New GATEWAY vectors for high throughput analyses of protein-protein interactions by bimolecular fluorescence complementation. Mol. Plant. 2009;2 doi: 10.1093/mp/ssp040. PubMed DOI

Clough S.J., Bent A.F. Floral dip: A simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana-Clough-1998-The Plant Journal-Wiley Online Library. Plant J. 2008;16:735–743. doi: 10.1046/j.1365-313x.1998.00343.x. PubMed DOI

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Nelson B.K., Cai X., Nebenführ A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. Cell Mol. Biol. 2007;51:1126–1136. doi: 10.1111/j.1365-313X.2007.03212.x. PubMed DOI

Dupl’áková N., Dobrev P.I., Reňák D., Honys D. Rapid separation of Arabidopsis male gametophyte developmental stages using a Percoll gradient. Nat. Protoc. 2016;11:1817–1832. doi: 10.1038/nprot.2016.107. PubMed DOI

Andrews S., Bitterncourt S. FastQC: A Quality Control Tool for High Throughput Sequence Data â“ ScienceOpen. [(accessed on 6 December 2019)]; Available online: http://www.bioinformatics.babraham.ac.uk/projects/fastqc.

Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. 2011;17 doi: 10.14806/ej.17.1.200. DOI

Babraham Bioinformatics. [(accessed on 21 June 2019)]; Available online: http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/

Dobin A., Davis C.A., Schlesinger F., Drenkow J., Zaleski C., Jha S., Batut P., Chaisson M., Gingeras T.R. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. doi: 10.1093/bioinformatics/bts635. PubMed DOI PMC

Liao Y., Smyth G.K., Shi W. featureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–930. doi: 10.1093/bioinformatics/btt656. PubMed DOI

Pimentel H., Bray N.L., Puente S., Melsted P., Pachter L. Differential analysis of RNA-seq incorporating quantification uncertainty. Nat. Methods. 2017;14:687–690. doi: 10.1038/nmeth.4324. PubMed DOI

Love M.I., Huber W., Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:1–21. doi: 10.1186/s13059-014-0550-8. PubMed DOI PMC

Mi H., Muruganujan A., Huang X., Ebert D., Mills C., Guo X., Thomas P.D. Protocol Update for large-scale genome and gene function analysis with the PANTHER classification system (v.14.0) Nat. Protoc. 2019;14:703–721. doi: 10.1038/s41596-019-0128-8. PubMed DOI PMC

Yamaguchi N., Winter C.M., Wu M.-F., Kwon C.S., William D.A., Wagner D. PROTOCOLS: Chromatin Immunoprecipitation from Arabidopsis Tissues. Arab. Book. 2014;12 doi: 10.1199/tab.0170. PubMed DOI PMC

Li H., Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2020;25:1754–1760. doi: 10.1093/bioinformatics/btp324. PubMed DOI PMC

Zhang Y., Liu T., Meyer C.A., Eeckhoute J., Johnson D.S., Bernstein B.E., Nusbaum C., Myers R.M., Brown M., Li W., et al. Model-based Analysis of ChIP-Seq (MACS) Genome Biol. 2008;9:1–9. doi: 10.1186/gb-2008-9-9-r137. PubMed DOI PMC

Ross-Innes C.S., Stark R., Teschendorff A.E., Holmes K.A., Ali H.R., Dunning M.J., Brown G.D., Gojis O., Ellis I.O., Green A.R., et al. Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature. 2012;481:389–393. doi: 10.1038/nature10730. PubMed DOI PMC

Yu G., Wang L.-G., He Q.-Y. ChIPseeker: An R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics. 2015;31 doi: 10.1093/bioinformatics/btv145. PubMed DOI

Hulsen T., Vlieg J.d., Alkema W. BioVenn—A web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. BMC Genom. 2008;9 doi: 10.1186/1471-2164-9-488. PubMed DOI PMC

Machanick P., Bailey T.L. MEME-ChIP: Motif analysis of large DNA datasets. Bioinformatics. 2011;27 doi: 10.1093/bioinformatics/btr189. PubMed DOI PMC

Ge S.X., Jung D., Yao R. ShinyGO: A graphical gene-set enrichment tool for animals and plants. Bioinformatics. 2020;36:2628–2629. doi: 10.1093/bioinformatics/btz931. PubMed DOI PMC

Afgan E., Baker D., Batut B., Beek M.v.d., Bouvier D., Cech M., Chilton J., Clements D., Coraor N., Grüning B.A., et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018;46:W537–W544. doi: 10.1093/nar/gky379. PubMed DOI PMC

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