BrPP5.2 Overexpression Confers Heat Shock Tolerance in Transgenic Brassica rapa through Inherent Chaperone Activity, Induced Glucosinolate Biosynthesis, and Differential Regulation of Abiotic Stress Response Genes
Abstract
:1. Introduction
2. Results
2.1. BrPP5.2 Expression Profiling in Different Tissues, Hormones, Stress Agents and Development of BrPP5.2-Overexpressing Transgenic Lines
2.2. BrPP5.2 Overexpression Results in Enhanced Heat Shock Stress Tolerance in Transgenic Lines
2.3. BrPP5.2 Possesses Holdase Chaperone Activity
2.4. Transcriptomic Signatures of BrPP5.2OX Lines
2.5. Chromosome-Wise Distribution of DEGs
2.6. BrPP5.2 Overexpression in Transgenic Lines Causes Changes in Metabolic and Abiotic Stress Responses
2.7. Stress-, Hormone-Response, and Development-Related Gene Expression Patterns
2.8. Expression Pattern of Phytohormone-Responsive DEGs
2.9. Expression Pattern of Photosynthesis-, Light-, and Circadian-Related Genes
2.10. Expression Profiling of Temperature Stimulus Response (TSR) DEGs at High Temperature Stress Conditions
2.11. Functional Classification of Significant DEGs
2.12. Changes in the Expression Pattern of GSL Biosynthesis-Related Genes and GSL Content in Transgenic Lines
2.13. Differential Splicing of Potential Targets
2.14. Brassica, Transgenic Specific DEGs
3. Discussion
BrPP5.2 Overexpression Confers Thermotolerance in Transgenic Lines Through Multiple Regulatory Mechanisms
4. Materials and Methods
4.1. Plant Materials and Stress Treatments
4.2. Expression Profiling of BrPP5.2 during Exogenous Application of Phytohormones and Stress Agents and Tissue-Specific Expression
4.3. Designing of the BrPP5.2 Overexpression Construct and Development of Transgenic B. rapa Lines
4.4. Phenotyping of BrPP5.2OX Lines for Heat Shock Stress Tolerance
4.5. Expression and Purification of Recombinant BrPP5.2 and Analysis of Holdase Chaperone Activity
4.6. Genome-Wide Analysis of Expression Changes of BrPP5.2 Downstream Genes by RNA-Seq
4.7. Gene Ontology, Functional Annotation, Statistical Enrichment, and Pathway Analyses
4.8. qRT-PCR-Based Relative Quantification of Temperature-Response DEGs in Heat Stress
4.9. Analysis of GSL Content in Transgenic and Control Seedlings under Optimal and High Temperature Stress Conditions
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- País, S.M.; Téllez-iñón, M.T.; Capiati, D.A. Serine/threonine protein phosphatases type 2A and their roles in stress signaling. Plant Signal. Behav. 2009, 4, 1013–1015. [Google Scholar] [CrossRef] [PubMed]
- Luan, S. Protein Phosphatases in Plants. Annu. Rev. Plant Biol. 2003, 54, 63–92. [Google Scholar] [CrossRef] [PubMed]
- Cohen, P.T.W. Overview of protein serine/threonine phosphatases. In Protein Phosphatases; Ariño, J.N., Alexander, D.R., Eds.; Springer: Berlin/Heidelberg, Germany, 2004; pp. 1–20. ISBN 978-3-540-40035-6. [Google Scholar]
- Farkas, I.; Dombrádi, V.; Miskei, M.; Szabados, L.; Koncz, C. Arabidopsis PPP family of serine/threonine phosphatases. Trends Plant Sci. 2007, 12, 169–176. [Google Scholar] [CrossRef] [Green Version]
- Bheri, M.; Pandey, G.K. Protein phosphatases meet reactive oxygen species in plant signaling networks. Environ. Exp. Bot. 2019, 161, 26–40. [Google Scholar] [CrossRef]
- Máthé, C.; Garda, T.; Freytag, C.; M-Hamvas, M. The Role of Serine-Threonine Protein Phosphatase PP2A in Plant Oxidative Stress Signaling—Facts and Hypotheses. Int. J. Mol. Sci. 2019, 20, 3028. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dai, M.; Xue, Q.; Mccray, T.; Margavage, K.; Chen, F.; Lee, J.H.; Nezames, C.D.; Guo, L.; Terzaghi, W.; Wan, J.; et al. The PP6 phosphatase regulates ABI5 phosphorylation and abscisic acid signaling in Arabidopsis. Plant Cell 2013, 25, 517–534. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bajsa, J.; Pan, Z.; Duke, S.O. Serine/threonine protein phosphatases: Multi-purpose enzymes in control of defense mechanisms. Plant Signal. Behav. 2011, 6, 1921–1925. [Google Scholar] [CrossRef] [Green Version]
- Kim, D.; Kang, J.; Yang, S.; Chung, K.; Song, P. A Phytochrome-Associated Protein Phosphatase 2A Modulates Light Signals in Flowering Time Control in Arabidopsis. Plant Cell 2002, 14, 3043–3056. [Google Scholar] [CrossRef] [Green Version]
- Ryu, J.S.; Kim, J.I.; Kunkel, T.; Kim, B.C.; Cho, D.S.; Hong, S.H.; Kim, S.H.; Fernández, A.P.; Kim, Y.; Alonso, J.M.; et al. Phytochrome-specific type 5 phosphatase controls light signal flux by enhancing phytochrome stability and affinity for a signal transducer. Cell 2005, 120, 395–406. [Google Scholar] [CrossRef] [Green Version]
- Uhrig, R.G.; Labandera, A.-M.; Moorhead, G.B. Arabidopsis PPP family of serine/threonine protein phosphatases: Many targets but few engines. Trends Plant Sci. 2013, 18, 505–513. [Google Scholar] [CrossRef]
- Hammond, R.K. The Protein Phosphatase 4 Complex Functions in miRNA Biogenesis in Arabidopsis. Plant Cell 2019, 31, 278–279. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Park, J.H.; Lee, S.Y.; Kim, W.Y.; Jung, Y.J.; Chae, H.B.; Jung, H.S.; Kang, C.H.; Shin, M.R.; Kim, S.Y.; Su’udi, M.; et al. Heat-induced chaperone activity of serine/threonine protein phosphatase 5 enhances thermotolerance in Arabidopsis thaliana. New Phytol. 2011, 191, 692–705. [Google Scholar] [CrossRef] [PubMed]
- de Dios Barajas-López, J.; Kremnev, D.; Shaikhali, J.; Piñas-Fernández, A.; Strand, Å. PAPP5 Is Involved in the Tetrapyrrole Mediated Plastid Signalling during Chloroplast Development. PLoS ONE 2013, 8, e60305. [Google Scholar] [CrossRef]
- Dai, M.; Zhang, C.; Kania, U.; Chen, F.; Xue, Q.; McCray, T.; Li, G.; Qin, G.; Wakeley, M.; Terzaghi, W.; et al. A PP6-type phosphatase holoenzyme directly regulates PIN phosphorylation and auxin efflux in Arabidopsis. Plant Cell 2012, 24, 2497–2514. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lillo, C.; Kataya, A.R.A.; Heidari, B.; Creighton, M.T.; Nemie-Feyissa, D.; Ginbot, Z.; Jonassen, E.M. Protein phosphatases PP2A, PP4 and PP6: Mediators and regulators in development and responses to environmental cues. Plant Cell Environ. 2014, 37, 2631–2648. [Google Scholar] [CrossRef] [PubMed]
- Møller, S.G.; Kim, Y.S.; Kunkel, T.; Chua, N.H. PP7 is a positive regulator of blue light signaling in Arabidopsis. Plant Cell 2003, 15, 1111–1119. [Google Scholar] [CrossRef] [Green Version]
- Liu, H.T.; Li, G.L.; Chang, H.; Sun, D.Y.; Zhou, R.G.; Li, B. Calmodulin-binding protein phosphatase PP7 is involved in thermotolerance in Arabidopsis. Plant Cell Environ. 2007, 30, 156–164. [Google Scholar] [CrossRef]
- Genoud, T.; Treviño Santa Cruz, M.; Kulisic, T.; Sparla, F.; Fankhauser, C.; Métraux, J.-P. The Protein Phosphatase 7 Regulates Phytochrome Signaling in Arabidopsis. PLoS ONE 2008, 3, e2699. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Shin, H.; Shin, H.S.; Guo, Z.; Blancaflor, E.B.; Masson, P.H.; Chen, R. Complex regulation of Arabidopsis AGR1/PIN2-mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases. Plant J. 2005, 42, 188–200. [Google Scholar] [CrossRef]
- Shi, Y. Serine/Threonine Phosphatases: Mechanism through Structure. Cell 2009, 139, 468–484. [Google Scholar] [CrossRef] [Green Version]
- Yang, J.; Roe, S.M.; Cliff, M.J.; Williams, M.A.; Ladbury, J.E.; Cohen, P.T.W.; Barford, D. Molecular basis for TPR domain-mediated regulation of protein phosphatase 5. EMBO J. 2005, 24, 1–10. [Google Scholar] [CrossRef] [Green Version]
- Li, G.; Zhang, C.; Zhang, G.; Fu, W.; Feng, B.; Chen, T.; Peng, S.; Tao, L.; Fu, G. Abscisic Acid Negatively Modulates Heat Tolerance in Rolled Leaf Rice by Increasing Leaf Temperature and Regulating Energy Homeostasis. Rice 2020, 13, 18. [Google Scholar] [CrossRef] [PubMed]
- Cheng, F.; Liu, S.; Wu, J.; Fang, L.; Sun, S.; Liu, B.; Li, P.; Hua, W.; Wang, X. BRAD, the genetics and genomics database for Brassica plants. BMC Plant Biol. 2011, 11, 136. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wang, X.; Wu, J.; Liang, J.; Cheng, F.; Wang, X. Brassica database (BRAD) version 2.0: Integrating and mining Brassicaceae species genomic resources. Database 2015, 2015, bav093. [Google Scholar] [CrossRef] [Green Version]
- He, Z.; Wu, J.; Sun, X.; Dai, M. The Maize Clade A PP2C Phosphatases Play Critical Roles in Multiple Abiotic Stress Responses. Int. J. Mol. Sci. 2019, 20, 3573. [Google Scholar] [CrossRef] [Green Version]
- Kang, H.; Kim, J.; Kim, B.; Jeong, H.; Hee, S.; Kyoung, E.; Lee, H.; Ok, P. Plant Science Overexpression of FTL1/DDF1, an AP2 transcription factor, enhances tolerance to cold, drought, and heat stresses in Arabidopsis thaliana. Plant Sci. 2011, 180, 634–641. [Google Scholar] [CrossRef]
- Guo, M.; Liu, J.-H.; Ma, X.; Luo, D.-X.; Gong, Z.-H.; Lu, M.-H. The Plant Heat Stress Transcription Factors (HSFs): Structure, Regulation, and Function in Response to Abiotic Stresses. Front. Plant Sci. 2016, 7, 114. [Google Scholar] [CrossRef] [Green Version]
- Ashraf, M.F.; Yang, S.; Wu, R.; Wang, Y.; Hussain, A.; Noman, A.; Khan, M.I.; Liu, Z.; Qiu, A.; Guan, D.; et al. Capsicum annuum HsfB2a Positively Regulates the Response to Ralstonia solanacearum Infection or High Temperature and High Humidity Forming Transcriptional Cascade with CaWRKY6 and CaWRKY40. Plant Cell Physiol. 2018, 59, 2608–2623. [Google Scholar] [CrossRef]
- Geng, X.; Zang, X.; Li, H.; Liu, Z.; Zhao, A.; Liu, J.; Peng, H.; Yao, Y.; Hu, Z.; Ni, Z.; et al. Unconventional splicing of wheat TabZIP60 confers heat tolerance in transgenic Arabidopsis. Plant Sci. 2018, 274, 252–260. [Google Scholar] [CrossRef] [PubMed]
- Khurana, N.; Sharma, N.; Khurana, P. Overexpression of a heat stress inducible, wheat myo-inositol-1-phosphate synthase 2 (TaMIPS2) confers tolerance to various abiotic stresses in Arabidopsis thaliana. Agri Gene 2017, 6, 24–30. [Google Scholar] [CrossRef]
- Yang, R.; Yu, G.; Li, H.; Li, X.; Mu, C. Overexpression of Small Heat Shock Protein LimHSP16.45 in Arabidopsis hsp17.6II Mutant Enhances Tolerance to Abiotic Stresses. Russ. J. Plant Physiol. 2020, 67, 231–241. [Google Scholar] [CrossRef]
- Bi, H.; Zhao, Y.; Li, H.; Liu, W. Wheat Heat Shock Factor TaHsfA6f Increases ABA Levels and Enhances Tolerance to Multiple Abiotic Stresses in Transgenic Plants. Int. J. Mol. Sci. 2020, 21, 3121. [Google Scholar] [CrossRef] [PubMed]
- Gai, W.X.; Ma, X.; Li, Y.; Xiao, J.J.; Khan, A.; Li, Q.H.; Gong, Z.H. CaHsfA1d Improves Plant Thermotolerance via Regulating the Expression of Stress- and Antioxidant-Related Genes. Int. J. Mol. Sci. 2020, 21, 8374. [Google Scholar] [CrossRef]
- Dickinson, P.J.; Kumar, M.; Martinho, C.; Yoo, S.J.; Lan, H.; Artavanis, G.; Charoensawan, V.; Schöttler, M.A.; Bock, R.; Jaeger, K.E.; et al. Chloroplast Signaling Gates Thermotolerance in Arabidopsis. Cell Rep. 2018, 22, 1657–1665. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lim, C.J.; Yang, K.A.; Hong, J.K.; Choi, J.S.; Yun, D.J.; Hong, J.C.; Chung, W.S.; Lee, S.Y.; Cho, M.J.; Lim, C.O. Gene expression profiles during heat acclimation in Arabidopsis thaliana suspension-culture cells. J. Plant Res. 2006, 119, 373–383. [Google Scholar] [CrossRef] [PubMed]
- Kamal, M.M.; Ishikawa, S.; Takahashi, F.; Suzuki, K.; Kamo, M.; Umezawa, T.; Shinozaki, K.; Kawamura, Y.; Uemura, M. Large-Scale Phosphoproteomic Study of Arabidopsis Membrane Proteins Reveals Early Signaling Events in Response to Cold. Int. J. Mol. Sci. 2020, 21, 8631. [Google Scholar] [CrossRef]
- Seo, M.S.; Jin, M.; Chun, J.H.; Kim, S.J.; Park, B.S.; Shon, S.H.; Kim, J.S. Functional analysis of three BrMYB28 transcription factors controlling the biosynthesis of glucosinolates in Brassica rapa. Plant Mol. Biol. 2016, 90, 503–516. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sánchez-Pujante, P.J.; Sabater-Jara, A.B.; Belchí-Navarro, S.; Pedreño, M.A.; Almagro, L. Increased Glucosinolate Production in Brassica oleracea var. italica Cell Cultures Due to Coronatine Activated Genes Involved in Glucosinolate Biosynthesis. J. Agric. Food Chem. 2019, 67, 102–111. [Google Scholar] [CrossRef] [PubMed]
- Abuyusuf, M.; Robin, A.H.K.; Kim, H.-T.; Islam, M.R.; Park, J.-I.; Nou, I.-S. Altered Glucosinolate Profiles and Expression of Glucosinolate Biosynthesis Genes in Ringspot-Resistant and Susceptible Cabbage Lines. Int. J. Mol. Sci. 2018, 19, 2833. [Google Scholar] [CrossRef] [Green Version]
- Zhang, J.; Wang, H.; Liu, Z.; Liang, J.; Wu, J.; Cheng, F.; Mei, S.; Wang, X. A naturally occurring variation in the BrMAM-3 gene is associated with aliphatic glucosinolate accumulation in Brassica rapa leaves. Hortic. Res. 2018, 5, 69. [Google Scholar] [CrossRef] [Green Version]
- Zang, Y.X.; Kim, J.H.; Park, Y.D.; Kim, D.H.; Hong, S.B. Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1. J. Biochem. Mol. Biol. 2008, 41, 472–478. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chhajed, S.; Mostafa, I.; He, Y.; Abou-Hashem, M.; El-Domiaty, M.; Chen, S. Glucosinolate Biosynthesis and the Glucosinolate–Myrosinase System in Plant Defense. Agronomy 2020, 10, 1786. [Google Scholar] [CrossRef]
- Wang, Y.; Wan, S.; Fan, H.; Yang, M.; Li, W.; Guan, R. A sulfotransferase gene BnSOT-like1 has a minor genetic effect on seed glucosinolate content in Brassica napus. Crop J. 2020, 8, 855–865. [Google Scholar] [CrossRef]
- del Carmen Martínez-Ballesta, M.; Moreno, D.; Carvajal, M. The Physiological Importance of Glucosinolates on Plant Response to Abiotic Stress in Brassica. Int. J. Mol. Sci. 2013, 14, 11607–11625. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Singh, D.; Singh, C.K.; Taunk, J.; Jadon, V.; Pal, M.; Gaikwad, K. Genome wide transcriptome analysis reveals vital role of heat responsive genes in regulatory mechanisms of lentil (Lens culinaris Medikus). Sci. Rep. 2019, 9, 12976. [Google Scholar] [CrossRef]
- Muthusamy, M.; Kim, J.Y.; Yoon, E.K.; Kim, J.A.; Lee, S.I. BrEXLB1, a Brassica rapa Expansin-Like B1 Gene Is Associated with Root Development, Drought Stress Response, and Seed Germination. Genes 2020, 11, 404. [Google Scholar] [CrossRef] [Green Version]
- Andrews, S. FastQC—A Quality Control Tool for High Throughput Sequence Data. Babraham Bioinforma. 2010. Available online: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 24 August 2020).
- Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [Green Version]
- Kim, D.; Paggi, J.M.; Park, C.; Bennett, C.; Salzberg, S.L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 2019, 37, 907–915. [Google Scholar] [CrossRef]
- 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. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Robinson, M.D.; McCarthy, D.J.; Smyth, G.K. edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010, 26, 139–140. [Google Scholar] [CrossRef] [Green Version]
- Love, M.I.; Huber, W.; Anders, S.; Huang, D.W.; Sherman, B.T.; Lempicki, R.A.; Robinson, M.D.; Mccarthy, D.J.; Smyth, G.K.; Liao, Y.; et al. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lun, A.T.L.; Chen, Y.; Smyth, G.K. It’s DE-licious: A recipe for differential expression analyses of RNA-seq experiments using quasi-likelihood methods in edgeR. In Methods in Molecular Biology; Humana Press Inc.: Totowa, NJ, USA, 2016; Volume 1418, pp. 391–416. [Google Scholar]
- Huang, D.W.; Sherman, B.T.; Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2009, 4, 44–57. [Google Scholar] [CrossRef]
- Huang, D.W.; Sherman, B.T.; Lempicki, R.A. Bioinformatics enrichment tools: Paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009, 37, 1–13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Raudvere, U.; Kolberg, L.; Kuzmin, I.; Arak, T.; Adler, P.; Peterson, H.; Vilo, J. g:Profiler: A web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 2019, 47, W191–W198. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lee, S.; Kim, J.; Lee, J. Differential glucosinolate profiles of radish leaves in response to postharvest drying treatment. Hortic. Environ. Biotechnol. 2021. [Google Scholar] [CrossRef]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Muthusamy, M.; Kim, J.H.; Kim, S.H.; Park, S.Y.; Lee, S.I. BrPP5.2 Overexpression Confers Heat Shock Tolerance in Transgenic Brassica rapa through Inherent Chaperone Activity, Induced Glucosinolate Biosynthesis, and Differential Regulation of Abiotic Stress Response Genes. Int. J. Mol. Sci. 2021, 22, 6437. https://doi.org/10.3390/ijms22126437
Muthusamy M, Kim JH, Kim SH, Park SY, Lee SI. BrPP5.2 Overexpression Confers Heat Shock Tolerance in Transgenic Brassica rapa through Inherent Chaperone Activity, Induced Glucosinolate Biosynthesis, and Differential Regulation of Abiotic Stress Response Genes. International Journal of Molecular Sciences. 2021; 22(12):6437. https://doi.org/10.3390/ijms22126437
Chicago/Turabian StyleMuthusamy, Muthusamy, Jong Hee Kim, Suk Hee Kim, So Young Park, and Soo In Lee. 2021. "BrPP5.2 Overexpression Confers Heat Shock Tolerance in Transgenic Brassica rapa through Inherent Chaperone Activity, Induced Glucosinolate Biosynthesis, and Differential Regulation of Abiotic Stress Response Genes" International Journal of Molecular Sciences 22, no. 12: 6437. https://doi.org/10.3390/ijms22126437