با همکاری مشترک دانشگاه پیام نور و انجمن بیوتکنولوژی جمهوری اسلامی ایران

نوع مقاله : علمی پژوهشی

نویسندگان

1 دانشجوی کارشناسی ارشد بیوتکنولوژی کشاورزی، گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه علوم کشاورزی و منابع طبیعی

2 استادیار، گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه علوم کشاورزی و منابع طبیعی خوزستان. ملاثانی. ایران.

3 استادیار، پژوهشکده آبزی پروری آب‌های جنوب کشور، موسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، اهواز، ایران

چکیده

گیاهان قادرند از طریق به خاطر سپردن تنش‌‌ گرمای قبلی (پرایمینگ)، نسبت به تنش‌‌های کشنده بعدی (برگشتی) تحمل گرمایی بدست آورند. اثر پرایمینگ که برای ساعت‌‌ها، روزها یا حتی نسل‌‌ها پس از تنش گرمایی برگشتی حفظ می‌‌شود، حافظه تنش گرمایی نامیده می‌‌شود. هدف از این مطالعه شناسایی ژن‌‌های کلیدی موثر در استقرار و تداوم حافظه تنش گرمایی است. در این مطالعه، داده‌‌های ریزآرایه پروفایل بیانی نمونه‌‌های آرابیدوپسیس از بانک داده‌‌های (Gene expression omnibus) GEO جمع‌‌آوری و ژن‌‌هایی با بیان افتراقی بر مبنای فعالیت بیشتر رونویسی‌‌شان تحت تنش برگشتی نسبت به تنش اول (مقایسه تیماری P+T/P) و همچنین القاء بیان پایدار تا 52 ساعت پس از فراغت از تنش (فاز حافظه) شناسایی شدند. سپس ژن‌‌های شناسایی شده به وسیله ابزارهای بیوانفورماتیک جهت دسته‌‌بندی هستی‌‌شناسی (Gene Ontology) و شبکه‌‌های برهم‌‌کنش پروتئینی (Protein-protein interaction networks) مورد بررسی قرار گرفتند. بررسی هستی‌‌شناسی عبارت‌‌ها نشان داد که ژن‌‌هایی با بیان افزایشی عمدتا با پاسخ سلولی و خوگیری به گرما و تاخوردگی پروتئین مرتبط بودند. از طریق خوشه‌‌بندی شبکه برهم‌‌کنش پروتئینی در عبارت‌‌ مربوط به "پاسخ به گرما "در مقایسه تیماری P+T/P، تعدادی از ژن‌‌های کلیدی موثر در استقرار حافظه تنش گرما نظیر HSP70T-2،HSP90 ، HSP60، AR192، HSP70، BIP2، J2، CLPB4، HOP3، HSP101، HSFA3، ROF1، HSFA2، HSP70B، CLPB3، MBF1C، FES1A شناسایی شدند. همچنین بر اساس تداوم بیان افتراقی ژن‌‌ها تا 52 ساعت پس از فراغت از تنش اول (فاز پرایمینگ) مشخص شد ژن‌‌هایی که در پایداری حافظه تنش گرما دخیل هستند عمدتا متعلق به اعضای خانواده پروتئین‌‌های شوک گرمایی کوچک (sHSPs) نظیر HSP17.6، HSP21، HSP17.6II، HAS32، HSP17.4، HSP18.2 و HSP22 بودند. علاوه بر این، در بررسی مسیرهای زیستی از طریق پایگاهKEGG (دانشنامه ژن‌‌ها و ژنوم‌‌های کیوتو) مشخص شد که ژن‌‌های حافظه تنش گرما عمدتا در مسیرهای پردازش پروتئین در شبکه اندوپلاسمی و فسفریلاسیون اکسیداتیو نقش داشتند. همچنین بررسی عناصر تنظیمی سیس در ناحیه پروموتری ژن‌‌های حافظه تنش نشان داد که خانواده فاکتورهای رونویسی bZIP، AP2;B3;RAV، MYB/SANT، HD-ZIP و GATA; tify دارای بیشترین جایگاه اتصال در ناحیه بالادست ژن‌‌های مذکور بودند. در مجموع این یافته‌‌ها اطلاعات مفیدی در خصوص آنالیز عملکردی و تنظیمی ژن‌‌های موثر در استقرار و تداوم حافظه تنش گرمایی و برهم کنش شبکه‌‌های پروتئینی آن‌‌ها ارائه داد که می‌‌توان از آنها در راستای بهبود ظرفیت تحمل گیاه تحت تنش شدید گرما استفاده کرد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Identification of key genes involved in the establishment and maintenance of heat stress memory in Arabidopsis seedlings using microarray data

نویسندگان [English]

  • Atena AlKian Abadi 1
  • Hengameh Taheri 2
  • Ayeh Sadat Sadr 3

1 M.Sc. in Agricultural biotechnology, Department of Plant Production and Genetics, Faculty of Agriculture, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran

2 Assistant Professor, Department of Plant Production and Genetics, Faculty of Agriculture, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran

3 Assistant Professor, South of Iran Aquaculture Research Institute (SIARI), Iranian Fisheries Science Research Institute, Agricultural Research Education and Extension Organization (AREEO), Ahvaz, Iran

چکیده [English]

Plants are able to acquire thermotolerance to the subsequent lethal stress through memorizing previous heat stress (HS) (Priming). A priming effect that can be sustained for several hours, days, or even generations after reverse heat stress, is called heat stress memory. The aim of this study was to identify effective key genes in establishing and maintaining heat stess memory. To achieve this, microarray data of the expression profile of Arabidopsis samples were retrieved from the GEO (Gene expression omnibus) database and differentially expressed genes (DEGs) were identified based on their higher transcriptional activation following recurring stress (in P+T/P treatment comparison) and their sustained induction even 52 hours after stress relief (during memory phase).The identified genes were further analyzed by bioinformatics tools for gene ontology (GO) classification and protein-protein interaction (PPI) networks. GO terms analysis disclosed that the up-regulated DEGs were mainly associated with cellular response to heat, heat acclimation and protein folding. By clustering of PPI networks in the term related to response to heat (in P+T/P treatment comparison), several candidate genes involved in thermomemory were identified including HSP70T-2, HSP91, AR192, HSP60, HSP70, BIP2, J2, CLPB4, HOP3, HSP101, ROF1, HSFA3, HSFA2, HSP70B, CLPB3, FES1A, MBF1C. Also, based on the sustained differential expression of genes even 52 hours after the priming phase, it was determined that genes responsible for maintaining heat stress memory were mainly members of the small heat shock protein family (sHSPs) such as HSP17.6, HSP21, HSP17.6II, HAS32, HSP17.4, HSP18.2 and HSP22. KEGG (Kyoto Encyclopedia of Genes and Genome) pathway analysis revealed that the HS memory genes were mainly involved in protein processing in the endoplasmic reticulum (ER) and oxidative phosphorylation. Furthermore, the analysis of cis-regulatory elements in the promoter regions of the thermomemory genes revealed that the transcription factors families of bZIP, AP2;B3;RAV, MYB/SANT, HD-ZIP and GATA; tify had the highest binding sites in their upstream regions. In summary, these findings provide useful information about functional and regulatory analysis of genes involved in the establishment and maintenance of heat stress memory, as well as their protein network interactions. This information can be used to improve the heat tolerance capacity of plants under extreme heat stress.

کلیدواژه‌ها [English]

  • Arabidopsis
  • Heat stress memory
  • Microarray
  • Protein interaction network
  • Thermopriming
Arif, M., Li, Z., Luo, Q., Li, L., Shen, Y., & Men, S. (2021). The BAG2 and BAG6 genes are involved in multiple abiotic stress tolerances in Arabidopsis thaliana. International Journal of Molecular Sciences, 22(11), 5856. Bäurle, I. (2016). Plant heat adaptation: priming in response to heat stress. F1000Research, 5. Bäurle, I. (2018, November). Can’t remember to forget you: chromatin-based priming of somatic stress responses. In Seminars in cell & developmental biology (Vol. 83, pp. 133-139). Academic Press. Bäurle, I. and Trindade, I. (2020). Chromatin regulation of somatic abiotic stress memory. Journal of Experimental Botany, 71(17): 5269-5279. Brzezinka, K., Altmann, S. and Bäurle, I. (2019). BRUSHY1/TONSOKU/MGOUN3 is required for heat stress memory. Plant, Cell & Environment, 42(3): 771-781. Carrigan, P.E., Sikkink, L.A., Smith, D.F. and Ramirez‐Alvarado, M. (2006). Domain: domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure. Protein Science, 15(3): 522-532. Charng, Y.Y., Liu, H.C., Liu, N.Y., Hsu, F.C. and Ko, S.S. (2006). Arabidopsis Hsa32, a novel heat shock protein, is essential for acquired thermotolerance during long recovery after acclimation. Plant Physiology, 140(4):1297-1305. dos Santos, T.B. and Vieira, L.G.E. (2020). Involvement of the galactinol synthase gene in abiotic and biotic stress responses: A review on current knowledge. Plant Gene, 24: 100258. Dragovic, Z., Shomura, Y., Tzvetkov, N., Hartl, F.U. and Bracher, A. (2006). Fes1p acts as a nucleotide exchange factor for the ribosome-associated molecular chaperone Ssb1p. Biological Chemistry, 387(12): 1593-1600. Gowda, N.K.C., Kaimal, J.M., Masser, A.E. Kang, W., Friedländer, M.R. and Andréasson, C. (2016). Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast. Molecular Biology of the Cell, 27(8): 1210-1219. Hall, D. (2020). On the nature of the optimal form of the holdase‐type chaperone stress response. FEBS Letters, 594(1): 43-66. Haslbeck, M., Weinkauf, S. and Buchner J. (2019). Small heat shock proteins: simplicity meets complexity. Journal of Biological Chemistry, 294(6): 2121-2132. Heidari, P., Ahmadizadeh, M. and NajafiZarrini, H. (2015). In Silico Analysis of Cis-regulatory Elements on co-expressed genes. Journal of Biological &. Environmental Sciences, 9(25): 1-9. Hilker, M., Schwachtje, J., Baier, M., Balazadeh, S., Bäurle, I., Geiselhardt, S., Hincha, D.K., Kunze, R., Mueller‐Roeber, B., Rillig, M.C. and Rolff, J. (2016). Priming and memory of stress responses in organisms lacking a nervous system. Biological Reviews, 91(4): 1118-1133. Jakoby, M., Weisshaar, B., Dröge-Laser, W., Vicente-Carbajosa, J., Tiedemann, J., Kroj, T. and Parcy, F. (2002). bZIP transcription factors in Arabidopsis. Trends in Plant Science, 7: 106–111 Khan, A., Khan, V., Pandey, K., Sopory, S.K. and Sanan-Mishra, N. (2022). Thermo-priming mediated cellular networks for abiotic stress management in plants. Frontiers in Plant Science, 13: 866409. Kushawaha, A.K, Khan, A., Sopory, S.K. and Sanan-Mishra, N. (2021). Priming by high temperature stress induces microRNA regulated heat shock modules indicating their involvement in thermopriming response in rice. Life (Basel), 11(4): 291. Lämke, J., Brzezinka, K., Altmann, S. and Bäurle, I. (2016a). A hit‐and‐run heat shock factor governs sustained histone methylation and transcriptional stress memory. EMBO Journal, 35(2): 162-175. Lämke, J., Brzezinka, K and Bäurle I. (2016b). HSFA2 orchestrates transcriptional dynamics after heat stress in Arabidopsis thaliana. Transcription, 7(4): 111–114 Lämke, J. and Bäurle, I. (2017). Epigenetic and chromatin-based mechanisms in environmental stress adaptation and stress memory in plants. Genome Biology, 18(1): 1-11. Lee, J. G. and Ye, Y. (2013). Bag6/Bat3/Scythe: A novel chaperone activity with diverse regulatory functions in protein biogenesis and degradation. Bioessays, 35: 377-385. Lin, M.Y., Chai, K.H., Ko, S.S., Kuang, L.Y., Lur, H.S. and Charng, Y.Y. (2014). A positive feedback loop between heat shock protein101 and heat stress-associated 32-kd protein modulates long-term acquired thermotolerance illustrating diverse heat stress responses in rice varieties. Plant Physiology, 164(4): 2045-2053. Ling, Y., Serrano, N., Gao, G., Atia, M., Mokhtar, M., Woo, Y.H., Bazin, J., Veluchamy, A., Benhamed, M. and Crespi, M. (2018). Thermopriming triggers splicing memory in Arabidopsis. Journal of Experimental Botany, 69(10): 2659-2675. Liu, Y., Zhang, A., Yin, H., Meng, Q., Yu, X., Huang, S., Wang, J., Ahmad, R., Liu, B. and Xu, Z.Y. (2018a). Trithorax‐group proteins ARABIDOPSIS TRITHORAX4 (ATX4) and ATX 5 function in abscisic acid and dehydration stress responses. New Phytologist, 217(4):1582-1597. Liu, H.C., Lämke, J., Lin, S.Y., Hung, M.J., Liu, K.M., Charng, Y.Y. and Bäurle, I. (2018b). Distinct heat shock factors and chromatin modifications mediate the organ‐autonomous transcriptional memory of heat stress. Plant Journal, 95(3): 401-413. Mayer, M. P. and Gierasch, L. M. (2019). Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones. Journal of Biological Chemistry, 294: 2085–2097. Meiri, D., Breiman, A. (2009). Arabidopsis ROF1 (FKBP62) modulates thermotolerance by interacting with HSP90. 1 and affecting the accumulation of HsfA2‐regulated sHSPs. Plant Journal, 59(3): 387-399. Mishra, R.C. and Grover, A. (2016). ClpB/Hsp100 proteins and heat stress tolerance in plants. Critical Reviews in Biotechnology, 36(5): 862-874. Mogk, A., Bukau, B. and Kampinga, H.H. (2018). Cellular handling of protein aggregates by disaggregation machines. Molecular Cell, 69(2): 214-226. Nollen, E.A. and Morimoto, R.I. (2002). Chaperoning signaling pathways: molecular chaperones as stress-sensing 'heat shock' proteins. Journal of Cell Science, 115(14): 2809-2816. Oberkofler, V., Pratx, L. and Bäurle, I. (2021). Epigenetic regulation of abiotic stress memory: maintaining the good things while they last. Current Opinion in Plant Biology, 61: 102007. Panikulangara, T.J., Eggers-Schumacher, G., Wunderlich, M., Stransky, H. and Schöffl, F. (2004). Galactinol synthase 1. A novel heat shock factor target gene responsible for heat-induced synthesis of raffinose family oligosaccharides in Arabidopsis. Plant Physiology, 136(2):3148-3158. Parcerisa, I.L., Rosano, G.L. and Ceccarelli, E.A. (2020). Biochemical characterization of ClpB3, a chloroplastic disaggregase from Arabidopsis thaliana. Plant Molecular Biology, 104(4): 451-465. Ritchie, ME., Phipson, B., Wu, D.I., Hu, Y., Law, C.W., Shi, W. and Smyth, G.K. (2015). limma powers diferential expression analyses for RNA sequencing and microarray studies. Nucleic Acids Research, 43(7):e47 Rolly, N.K., Imran, Q.M., Shahid, M., Imran, M., Khan, M., Lee, S.U., Hussain, A., Lee, I.J. and Yun, B.W. (2020). Drought-induced AtbZIP62 transcription factor regulates drought stress response in Arabidopsis. Plant Physiology and Biochemistry, 156; 384–395. Sauer, R.T. and Baker, T.A. (2011). AAA+ proteases: ATP-fueled machines of protein destruction. Annual Review of Biochemistry, 80: 587-612. Sedaghatmehr, M. Mueller-Roeber, B. and Balazadeh, S. (2016). The plastid metalloprotease FtsH6 and small heat shock protein HSP21 jointly regulate thermomemory in Arabidopsis. Nature Communications, 7: 12439. Sequeira-Mendes, J., Aragüez, I., Peiró, R. Mendez-Giraldez, R., Zhang, X., Jacobsen, S.E., Bastolla, U. and Gutierrez, C. (2014). The functional topography of the Arabidopsis genome is organized in a reduced number of linear motifs of chromatin states. Plant Cell, 26(6): 2351-2366. Standaert, R. F., Galat, A., Verdine, G. L. and Schreiber, S. L. (1990). Molecular cloning and overexpression of the human FK506-binding protein FKBP. Nature, 346(6285): 671-674 Stief, A., Altmann, S., Hoffmann, K. et al. (2014a). Arabidopsis miR156 regulates tolerance to recurring environmental stress through SPL transcription factors. Plant Cell, 26(4) :1792-1807. Stief, A., Brzezinka, K., Lämke, J. and Bäurle, I. (2014b). Epigenetic responses to heat stress at different time scales and the involvement of small RNAs. Plant signaling & behavior, 9(10): e970430. Wang, W., Vinocur, B., Shoseyov, O. and Altman, A. (2004). Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends in Plant Science, 9(5): 244-252. Wu, T.Y., Juan, Y.T., Hsu, Y.H., Wu, S.H., Liao, H.T., Fung, R.W. and Charng, Y.Y. (2013). Interplay between heat shock proteins HSP101 and HSA32 prolongs heat acclimation memory posttranscriptionally in Arabidopsis.Plant Physiology,161(4):2075–2084. Yamaguchi, N. (2021). Heat memory in plants: histone modifications, nucleosome positioning and miRNA accumulation alter heat memory gene expression. Genes & Genetic Systems, 96(5): 229-235. Yamaguchi, N., Matsubara, S., Yoshimizu ,K., Seki, M., Hamada, K., Kamitani, M., Kurita, Y., Nomura, Y., Nagashima, K., Inagaki, S., Suzuki, T., Gan, E.S., To, T., Kakutani, T., Nagano, A.J., Satake, A. and Ito, T. (2021). H3K27me3 demethylases alter HSP22 and HSP17. 6C expression in response to recurring heat in Arabidopsis. Nature Communications, 12(1): 1-16. Yeh, C.H., Kaplinsky, N.J., Hu, C., Charng, Y.Y. (2012). Some like it hot, some like it warm: phenotyping to explore thermotolerance diversity. Plant Science, 195: 10-23. Zhang, X., Bernatavichute, Y.V., Cokus, S, Pellegrini, M., and Jacobsen, S.E. (2009). Genome-wide analysis of mono-, di- and trimethylation of histone H3 lysine 4 in Arabidopsis thaliana. Genome Biology, 10: 1-14. Zhang, J.X., Wang, C., Yang, C.Y. Wang, J.Y., Chen, L., Bao, X.M., Zhao, Y.X., Zhang, H. and Liu, J. (2010). The role of Arabidopsis AtFes1A in cytosolic Hsp70 stability and abiotic stress tolerance. Plant Journal, 62(4): 539-548. Zhang, B., Feng, C., Chen, L., Li, B., Zhang, X. and Yang X. (2022). Identification and functional analysis of bZIP genes in cotton response to drought stress. International Journal of Molecular Sciences, 28: 23(23):14894. Zhou, Y., Wang, Y., Xu, F. and Song, C. (2022). Small HSPs play an important role in crosstalk between HSF-HSP and ROS pathways in heat stress response through transcriptomic analysis in lilies (Lilium longiflorum). BMC Plant Biology, 22(1): 202. Zhuang, Y., Wei, M., Ling, C., Liu, Y., Amin, A. K., Li, P., ... & Wang, S. (2021). EGY3 mediates chloroplastic ROS homeostasis and promotes retrograde signaling in response to salt stress in Arabidopsis. Cell Reports, 36(2), 109384. Zuiderweg, E. R., Hightower, L. E., & Gestwicki, J. E. (2017). The remarkable multivalency of the Hsp70 chaperones. Cell Stress and Chaperones, 22, 173-189.