Identification of EgrZFP-EARs of the C2H2 Zinc Finger Protein Gene Family in Eucalyptus grandis and Analysis of Their Expression Pattern Under Abiotic Stresses
YU Jian-Feng, HONG Jia-Du, ZHAO Shuang, LI Xiao-Mei, WEN Dan-Dan, CHENG Long-Jun*
College of Forestry and Biotechnology/State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China
Abstract：Eucalyptus spp. are widely planted in south of China as a fast-growing timber species. However, the resistance to abiotic stresses such as low temperature, drought, and salinity of them is very poor. Zinc finger protein (ZPF) transcription factor members containing the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif have been shown to be extensively involved in plant responses to abiotic stresses such as cold, high temperature, drought and salinity. Based on the genome-wide identification of Eucalyptus grandis C2H2-type ZFP, the EgrZFP-EAR genes with EAR motif was screened. The evolutionary relationships of EgrZFP-EAR, the types, numbers and distributions of EAR motifs in their proteins, chromosome localization and gene replication of them, as well as gene and protein sequence structure and the cis-elements on their promoters were analyzed. In addition, transcriptome data and qRT-PCR were used to analyze the expression patterns of EgrZFP-EAR in different tissues and 4 ℃, high temperature (42 ℃), drought, NaCl (300 mmol/L), ABA (100 μmol/L) and MeJA (100 μmol/L) treatments of E. grandis seedlings. The results showed that there were 118 C2H2-type ZFP in E. grandis, 57 of which were EgrZFP-EAR members, and 54 of these members encoded ZFP proteins containing Q-type sequence (QALGGH). They contained 3 types of EAR motifs: LxLxL, DLNx (x) p, and their overlap. The LxLxL type had the largest number of them, with a total of 98. Evolutionarily, genome-wide replication, tandem duplication and segment duplication all played roles in the expansion of these genes. Stress responsive elements such as DRE, MBS, LTR and STRE, and hormone responsive elements including ABRE and CGTCA-motif were distributed on the EgrZFP-EAR promoters. Tissue expression patterns were divided into 4 types: high expression in shoot tips and young leaves; high expression in phloem; high expression in mature xylem; and high expression in phloem, immature and mature xylem. The expression patterns of EgrZFP-EAR gene in leaves of E. grandis seedlings treated with 4 ℃ for different time (2, 6, 12, 24, 48 h) were classified into 3 types. Genes in the first type were induced persistently; the expression of genes in the second type ascended firstly and then descended; and in the third type, genes' expression was mainly inhibited. The selected 12 genes from different EgrZFP-EAR subgroup were also responsive differently to high temperature, drought, NaCl, ABA and MeJA treatments, implicating the complex function of EgrZFP-EAR gene under abiotic stresses. The results provide an foundation for further revealing the molecular mechanism of stress resistance, the exploitation of stress-resistant gene resources and molecular-assisted stress resistance breeding of Eucalyptus.
俞键烽, 洪家都, 赵爽, 李笑眉, 温丹丹, 程龙军. 巨桉C2H2锌指蛋白基因家族EgrZFP-EARs鉴定及其在非生物逆境下的表达模式分析[J]. 农业生物技术学报, 2024, 32(8): 1792-1808.
YU Jian-Feng, HONG Jia-Du, ZHAO Shuang, LI Xiao-Mei, WEN Dan-Dan, CHENG Long-Jun. Identification of EgrZFP-EARs of the C2H2 Zinc Finger Protein Gene Family in Eucalyptus grandis and Analysis of Their Expression Pattern Under Abiotic Stresses. 农业生物技术学报, 2024, 32(8): 1792-1808.
[1] 杨宁, 从青, 王晓荣, 等. 2019. 巨桉EgrBBX基因家族鉴定及其在非生物逆境处理下的表达分析[J]. 农业生物技术学报, 28(4): 658-671. (Yang N, Cong Q,Wang X R, et al.2019. Identification of EgrBBX gene family and its expression analysis under abiotic stress in Eucalyptus grandis[J]. Journal of Agricultural Biotechnology, 28(4): 658-671.) [2] Arrey-Salas O, Caris-Maldonado J C, Hernández-Rojas B, et al.2021. Comprehensive genome-wide exploration of c2h2 zinc finger family in grapevine (Vitis vinifera L.): Insights into the roles in the pollen development regulation[J]. Genes, 12(2): 302. [3] Berg J M, Shi Y.1996. The galvanization of biology: a growing appreciation for the roles of zinc[J]. Science, 271(5252): 1081-1085. [4] Chow V, Kirzinger M W, Kagale S.2023. Lend me your EARs: A systematic review of the broad functions of EAR motif-containing transcriptional repressors in plants[J]. Genes, 14(2): 270. [5] Davletova S, Schlauch K, Coutu J, et al.2005. The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in Arabidopsis[J]. Plant Physiology, 139(2): 847-856. [6] Englbrecht C C, Schoof H, Böhm S.2004. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome[J]. BMC Genomics, 5: 39. [7] Gourcilleau D, Lenne C, Armenise C, et al.2011. Phylogenetic study of plant Q-type C2H2 zinc finger proteins and expression analysis of poplar genes in response to osmotic, cold and mechanical stresses[J]. DNA Research, 18(2): 77-92. [8] Han G, Lu C, Guo J, et al.2020. C2H2 zinc finger proteins: Master regulators of abiotic stress responses in plants[J]. Frontiers in Plant Science, 11: 115. [9] Hiratsu K, Matsui K, Koyama T, et al.2003. Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis[J]. The Plant Journal, 34(5): 733-739. [10] Huang J, Sun S J, Xu D Q, et al.2009. Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245[J]. Biochemical and Biophysical Research Communications, 389(3): 556-561. [11] Jiao Z, Wang L, Du H, et al.2020. Genome-wide study of C2H2 zinc finger gene family in Medicago truncatula[J]. BMC Plant Biology, 20: 1-17. [12] Kagale S, Links M G, Rozwadowski K.2010. Genome-wide analysis of ethylene-responsive element binding factor-associated amphiphilic repression motif-containing transcriptional regulators in Arabidopsis[J]. Plant Physiology, 152(3): 1109-1134. [13] Kagale S, Rozwadowski K.2011. EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression[J]. Epigenetics, 6(2): 141-146. [14] Kazan K.2006. Negative regulation of defence and stress genes by EAR-motif-containing repressors[J]. Trends in Plant Science, 11(3): 109-112. [15] Kiełbowicz-Matuk A.2012. Involvement of plant C2H2-type zinc finger transcription factors in stress responses[J]. Plant Science, 185(4): 78-85. [16] Kim J C, Lee S H, Cheong Y H, et al.2001. A novel cold‐inducible zinc finger protein from soybean, SCOF‐1, enhances cold tolerance in transgenic plants[J]. The Plant Journal, 25(3): 247-259. [17] Kubo K-i, Sakamoto A, Kobayashi A, et al.1998. Cys2/His2 zinc-finger protein family of Petunia: Evolution and general mechanism of target-sequence recognition[J]. Nucleic Acids Research, 26(2): 608-615. [18] Laity J H, Lee B M, Wright P E.2001. Zinc finger proteins: New insights into structural and functional diversity[J]. Current Opinion in Structural Biology, 11(1): 39-46. [19] Lawrence S D, Novak N G.2015. Mining the Brassica oleracea genome for Q-type C2H2 zinc finger transcription factor genes[J]. Plant Molecular Biology Reporter, 33: 1611-1617. [20] Liu Y, Khan A R, Azhar W, et al.2022a. Cys2/His2-type zinc finger proteins regulate plant growth and development[J]. Critical Reviews in Plant Sciences, 41(5): 351-363. [21] Liu Y, Khan A R, Gan Y.2022b. C2H2 zinc finger proteins response to abiotic stress in plants[J]. International Journal of Molecular Sciences, 23(5): 2730. [22] Mittler R, Kim Y, Song L, et al.2006. Gain-and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress[J]. FEBS Letters, 580(28-29): 6537-6542. [23] Pu J, Li M, Mao P, et al.2021. Genome-wide identification of the Q-type C2H2 transcription factor family in alfalfa(Medicago sativa) and expression analysis under different abiotic stresses[J]. Genes, 12(12): 1906. [24] Ouyang L, Arnold R J, Chen S, et al.2022. Prediction of the suitable distribution of Eucalyptus grandis in China and its responses to climate change[J]. New Forests, 53(3): 81-99. [25] Sakamoto H, Maruyama K, Sakuma Y, et al.2004. Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions[J]. Plant Physiology, 136(1): 2734-2746. [26] Schuh R, Aicher W, Gaul U, et al.1986. A conserved family of nuclear proteins containing structural elements of the finger protein encoded by Krüppel, a Drosophila segmentation gene[J]. Cell, 47(6): 1025-1032. [27] Sugano S, Kaminaka H, Rybka Z, et al.2003. Stress‐responsive zinc finger gene ZPT2‐3 plays a role in drought tolerance in Petunia[J]. The Plant Journal, 36(6): 830-841. [28] Sun S J, Guo S Q, Yang X, et al.2010. Functional analysis of a novel Cys2/His2-type zinc finger protein involved in salt tolerance in rice[J]. Journal of Experimental Botany, 61(10): 2807-2818. [29] Sun Z, Liu R, Guo B, et al.2019. Ectopic expression of GmZAT4, a putative C2H2-type zinc finger protein, enhances PEG and NaCl stress tolerances in Arabidopsis thaliana[J]. 3 Biotech, 9: 166. [30] Takatsuji H.1998. Zinc-finger transcription factors in plants[J]. Cellular and Molecular Life Sciences CMLS, 54: 582-596. [31] Takatsuji H.1999. Zinc-finger proteins: The classical zinc finger emerges in contemporary plant science[J]. Plant Molecular Biology, 39(4): 1073-1078. [32] Vogel J T, Zarka D G, Van Buskirk H A, et al.2005. Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis[J]. The Plant Journal, 41(2): 195-211. [33] Wang K, Ding Y, Cai C, et al.2019. The role of C2H2 zinc finger proteins in plant responses to abiotic stresses[J]. Physiologia Plantarum, 165(4): 690-700. [34] Wang S, Wei X, Cheng L, et al.2014. Identification of a C2H2-type zinc finger gene family from Eucalyptus grandis and its response to various abiotic stresses[J]. Biologia Plantarum, 58(2): 385-390. [35] Xie M, Sun J, Gong D, et al.2019. The roles of Arabidopsis C1-2i subclass of C2H2-type zinc-finger transcription factors[J]. Genes, 10(9): 653. [36] Yang J, Liu Y, Yan H, et al.2018. PlantEAR: Functional analysis platform for plant EAR motif-containing proteins[J]. Frontiers in Genetics, 9: 590. [37] Zhang X, Guo Q, Qin L, et al.2022. A Cys2His2 zinc finger transcription factor BpSZA1 positively modulates salt stress in Betula platyphylla[J]. Frontiers in Plant Science, 13: 823547. [38] Zhuang H, Wang H L, Zhang T, et al.2020. NONSTOP GLUMES1 encodes a C2H2 zinc finger protein that regulates spikelet development in rice[J]. The Plant Cell, 32(2): 392-413.
