OVEREXPRESSION OF <i>PTRLEA7</i>, A LATE EMBRYOGENESIS ABUNDANT FAMILY GENE FROM <i>PONCIRUS TRIFOLIATA</i>, CONFERS ENHANCED DROUGHT TOLERANCE BY ENHANCING ANTIOXIDANT CAPACITY

doi:10.15302/J-FASE-2020368

Front. Agr. Sci. Eng.

2021, Vol. 8

Issue (2) : 236-246 https://doi.org/10.15302/J-FASE-2020368

RESEARCH ARTICLE

OVEREXPRESSION OF PTRLEA7, A LATE EMBRYOGENESIS ABUNDANT FAMILY GENE FROM PONCIRUS TRIFOLIATA, CONFERS ENHANCED DROUGHT TOLERANCE BY ENHANCING ANTIOXIDANT CAPACITY

Tonglu WEI^1,², Dalong GUO², Jihong LIU¹(

)

¹. Key Laboratory of Horticultural Plant Biology (MOE), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China.
². College of Forestry, Henan University of Science and Technology, Luoyang 471023, China.

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Abstract

• A LEA family gene (PtrLEA7)was cloned from Poncirus trifoliata.

• PtrLEA7was strongly induced by stresses and ABA.

• PtrLEA7played a positive role in modulation of drought tolerance.

• Overexpression of PtrLEA7elevated antioxidant capacity.

Late embryogenesis abundant (LEA) genes encode highly hydrophilic proteins that are essential in abiotic stress responses. However, most LEA genes in higher plants have not yet been investigated. This study identified an LEA family gene (PtrLEA7) from Poncirus trifoliata and studied its function in drought tolerance. The full-length coding sequence of PtrLEA7 was 420 bp encoding a protein of 139 amino acids. Phylogenetic analysis shows that PtrLEA7 protein belongs to the LEA_4 subfamily. Expression profiling by qPCR found that PtrLEA7 was strongly induced by dehydration, cold and ABA treatments, and slightly induced by salt stress. Subcellular localization reveals that PtrLEA7 protein was located in both cytoplasm and nucleus. To investigate its function, transgenic plants of both tobacco and Poncirus trifoliata overexpressing PtrLEA7 were obtained. Stress tolerance assays show that overexpression lines had enhanced dehydration and drought tolerance compared with wild type plants, indicating that PtrLEA7 positively regulates drought tolerance. In addition, transgenic plants had much higher expression levels of three antioxidant enzyme genes (CAT, SOD and POD) and significantly increased catalase enzyme activity, accompanied by reduced reactive oxygen species accumulation in comparison with wild type plants. Collectively, this study demonstrates that PtrLEA7 can confer enhanced drought tolerance partially via enhancing antioxidant capacity.

Keywords abiotic stress antioxidant drought late embryogenesis abundant Poncirus trifoliata

Corresponding Author(s): Jihong LIU

Just Accepted Date: 17 November 2020 Online First Date: 04 January 2021 Issue Date: 13 July 2021

Cite this article:

Tonglu WEI,Dalong GUO,Jihong LIU. OVEREXPRESSION OF PTRLEA7, A LATE EMBRYOGENESIS ABUNDANT FAMILY GENE FROM PONCIRUS TRIFOLIATA, CONFERS ENHANCED DROUGHT TOLERANCE BY ENHANCING ANTIOXIDANT CAPACITY[J]. Front. Agr. Sci. Eng. , 2021, 8(2): 236-246.

URL:

https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2020368
https://academic.hep.com.cn/fase/EN/Y2021/V8/I2/236

Fig.1 Phylogenetic analysis of LEAs. Phylogenetic tree is constructed based on LEA protein sequences of Arabidopsis thaliana, Citrus sinensis and PtrLEA7. Different subfamilies are shown with different colors. The LEAs are divided into 10 subfamilies based on the taxonomy proposed in previous reports^[⁴^,⁹^]. The scale bar indicates the relative amount of change along branches.

Fig.2 Expression profiling of PtrLEA7 under abiotic stresses and ABA treatment. Relative expression levels are measured by qPCR at each time point of different treatments comprising dehydration (a), low temperature (b), salt (c), and ABA (d).

Fig.3 Subcellular localization of PtrLEA7. The upper and lower images indicate the fluorescence of the control (YFP, a–d) and the constructed vector (PtrLEA7-YFP, e–h), respectively. The images show representative cells under UV field (a, e), autofluorescence (b, f), bright-field (c, g), and merged field (d, h). Bar= 25 mm.

Fig.4 Drought and dehydration tolerance assays of transgenic tobacco plants overexpressing PtrLEA7. (a–c) Phenotype (a), chlorophyll fluorescence (b), and corresponding Fv/Fm values (c) of transgenic and wild type (WT) plants before and after drought treatment. (d–f) Phenotype (d), relative water loss (e), and electrolyte leakage (f) of transgenic and wild plants during or after dehydration treatment. Lines T4 and T8 are transgenic.

Fig.5 Dehydration tolerance assays of transgenic trifoliate orange plants overexpressing PtrLEA7. (a–c) Phenotype (a), chlorophyll fluorescence (b), and corresponding Fv/Fm values (c) of transgenic and wild type (WT) plants before and after dehydration treatment. (d) Relative water loss over a 48-h dehydration treatment. OE5 and OE8 represent two transgenic lines.

Fig.6 Investigations of antioxidant capacity and ROS accumulation in PtrLEA7-overexpressed trifoliate orange plants. (a,b) Relative expression levels of three antioxidant enzyme genes (CAT, SOD and POD) (a) and catalase (CAT) enzyme activity (b) in WT and two transgenic lines. (c) In situ ROS accumulation in the leaves of WT and two transgenic lines after dehydration treatment by 3,3′-diaminobenzidine and nitrotetrazolium blue chloride staining.

1	K Shinozaki, K Yamaguchi-Shinozaki. Gene networks involved in drought stress response and tolerance. Journal of Experimental Botany, 2007, 58(2): 221–227 https://doi.org/10.1093/jxb/erl164 pmid: 17075077
2	Z Huang, X J Zhong, J He, S H Jin, H D Guo, X F Yu, Y J Zhou, X Li, M D Ma, Q B Chen, H Long. Genome-wide identification, characterization, and stress-responsive expression profiling of genes encoding LEA (Late Embryogenesis Abundant) proteins in Moso bamboo (Phyllostachys edulis). PLoS One, 2016, 11(11): e0165953 pmid: 27829056
3	Y C Altunoglu, P Baloglu, E N Yer, S Pekol, M C Baloglu. Identification and expression analysis of LEA gene family members in cucumber genome. Plant Growth Regulation, 2016, 80: 225–241
4	M Hundertmark, D K Hincha. LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics, 2008, 9(1): 118 pmid: 18318901
5	J Grelet, A Benamar, E Teyssier, M H Avelange-Macherel, D Grunwald, D Macherel. Identification in pea seed mitochondria of a late-embryogenesis abundant protein able to protect enzymes from drying. Plant Physiology, 2005, 137(1): 157–167 https://doi.org/10.1104/pp.104.052480 pmid: 15618423
6	M J Wise, A Tunnacliffe. POPP the question: what do LEA proteins do? Trends in Plant Science, 2004, 9(1): 13–17 https://doi.org/10.1016/j.tplants.2003.10.012 pmid: 14729214
7	R O Magwanga, P Lu, J N Kirungu, H Lu, X Wang, X Cai, Z Zhou, Z Zhang, H Salih, K Wang, F Liu. Characterization of the late embryogenesis abundant (LEA) proteins family and their role in drought stress tolerance in upland cotton. BMC Genetics, 2018, 19(1): 6 https://doi.org/10.1186/s12863-017-0596-1 pmid: 29334890
8	Y Chen, C Li, B Zhang, J Yi, Y Yang, C Kong, C Lei, M Gong. The role of the late embryogenesis-abundant (LEA) protein family in development and the abiotic stress response: a comprehensive expression analysis of potato (Solanum tuberosum). Genes, 2019, 10(2): 148 https://doi.org/10.3390/genes10020148 pmid: 30781418
9	A M Pedrosa, C P S Martins, L P Gonçalves, M G C Costa. Late embryogenesis abundant (LEA) constitutes a large and diverse family of proteins involved in development and abiotic stress responses in sweet orange (Citrus sinensis L. Osb.). PLoS One, 2015, 10(12): e0145785 https://doi.org/10.1371/journal.pone.0145785 pmid: 26700652
10	B Xiao, Y Huang, N Tang, L Xiong. Over-expression of a LEA gene in rice improves drought resistance under the field conditions. Theoretical and Applied Genetics, 2007, 115(1): 35–46 https://doi.org/10.1007/s00122-007-0538-9 pmid: 17426956
11	Y Liang, K Kang, L Gan, S Ning, J Xiong, S Song, L Xi, S Lai, Y Yin, J Gu, J Xiang, S Li, B Wang, M Li. Drought-responsive genes, late embryogenesis abundant group3 (LEA3) and vicinal oxygen chelate, function in lipid accumulation in Brassica napus and Arabidopsis mainly via enhancing photosynthetic efficiency and reducing ROS. Plant Biotechnology Journal, 2019, 17(11): 2123–2142 https://doi.org/10.1111/pbi.13127 pmid: 30972883
12	Y Liu, L Wang, X Xing, L Sun, J Pan, X Kong, M Zhang, D Li. ZmLEA3, a multifunctional group 3 LEA protein from maize (Zea mays L.), is involved in biotic and abiotic stresses. Plant & Cell Physiology, 2013, 54(6): 944–959 https://doi.org/10.1093/pcp/pct047 pmid: 23543751
13	Y Liu, J Liang, L Sun, X Yang, D Li. Group 3 LEA protein, ZmLEA3, is involved in protection from low temperature stress. Frontiers of Plant Science, 2016, 7: 1011 https://doi.org/10.3389/fpls.2016.01011 pmid: 27471509
14	R Chandra Babu, J Zhang, A Blum, T H David Ho, R Wu, H T Nguyen. HVA1, a LEA gene from barley confers dehydration tolerance in transgenic rice (Oryza sativa L.) via cell membrane protection. Plant Science, 2004, 166(4): 855–862 https://doi.org/10.1016/j.plantsci.2003.11.023
15	A Candat, G Paszkiewicz, M Neveu, R Gautier, D C Logan, M H Avelange-Macherel, D Macherel. The ubiquitous distribution of late embryogenesis abundant proteins across cell compartments in Arabidopsis offers tailored protection against abiotic stress. Plant Cell, 2014, 26(7): 3148–3166 https://doi.org/10.1105/tpc.114.127316 pmid: 25005920
16	C Krüger, O Berkowitz, U W Stephan, R Hell. A metal-binding member of the late embryogenesis abundant protein family transports iron in the phloem of Ricinus communis L. Journal of Biological Chemistry, 2002, 277(28): 25062–25069 https://doi.org/10.1074/jbc.M201896200 pmid: 11983700
17	T Wei, Y Wang, Z Xie, D Guo, C Chen, Q Fan, X Deng, J H Liu. Enhanced ROS scavenging and sugar accumulation contribute to drought tolerance of naturally occurring autotetraploids in Poncirus trifoliata. Plant Biotechnology Journal, 2019, 17(7): 1394–1407 https://doi.org/10.1111/pbi.13064 pmid: 30578709
18	M Walter, C Chaban, K Schütze, O Batistic, K Weckermann, C Näke, D Blazevic, C Grefen, K Schumacher, C Oecking, K Harter, J Kudla. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant Journal, 2004, 40(3): 428–438 https://doi.org/10.1111/j.1365-313X.2004.02219.x pmid: 15469500
19	T Murashige, F Skoog. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 1962, 15(3): 473–497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
20	T Murashige, D P H Tucker. Growth factor requirements of citrus tissue culture. In: Chapman H D, ed. Proceedings of first international citrus symposium. Riverside, CA: University of California Press, 1969, 1155–1161
21	R B Horsch, J E Fry, N L Hoffmann, D Eichholtz, S G Rogers, R T Fraley. A simple and general method for transferring genes into plants. Science, 1985, 227(4691): 1229–1231 https://doi.org/10.1126/science.227.4691.1229 pmid: 17757866
22	X Z Fu, C W Chen, Y Wang, J H Liu, T Moriguchi. Ectopic expression of MdSPDS1 in sweet orange (Citrus sinensis Osbeck) reduces canker susceptibility: involvement of H2O2 production and transcriptional alteration. BMC Plant Biology, 2011, 11(1): 55 https://doi.org/10.1186/1471-2229-11-55 pmid: 21439092
23	B Dahro, F Wang, T Peng, J H Liu. PtrA/NINV, an alkaline/neutral invertase gene of Poncirus trifoliata, confers enhanced tolerance to multiple abiotic stresses by modulating ROS levels and maintaining photosynthetic efficiency. BMC Plant Biology, 2016, 16(1): 76 https://doi.org/10.1186/s12870-016-0761-0 pmid: 27025596
24	J Geng, J H Liu. The transcription factor CsbHLH18 of sweet orange functions in modulation of cold tolerance and homeostasis of reactive oxygen species by regulating the antioxidant gene. Journal of Experimental Botany, 2018, 69(10): 2677–2692 https://doi.org/10.1093/jxb/ery065 pmid: 29474667
25	J K Zhu. Abiotic stress signaling and responses in plants. Cell, 2016, 167(2): 313–324 https://doi.org/10.1016/j.cell.2016.08.029 pmid: 27716505
26	K K Nadarajah. ROS homeostasis in abiotic stress tolerance in plants. International Journal of Molecular Sciences, 2020, 21(15): 5208 https://doi.org/10.3390/ijms21155208 pmid: 32717820
27	S C Hand, M A Menze, M Toner, L Boswell, D Moore. LEA proteins during water stress: not just for plants anymore. Annual Review of Physiology, 2011, 73(1): 115–134 https://doi.org/10.1146/annurev-physiol-012110-142203 pmid: 21034219
28	Y T Ke, C A Lu, S J Wu, C H Yeh. Characterization of rice group 3 LEA genes in developmental stages and under abiotic stress. Plant Molecular Biology Reporter, 2016, 34(5): 1003–1015 https://doi.org/10.1007/s11105-016-0983-1
29	K S Wilhelm, M F Thomashow. Arabidopsis thaliana cor15b, an apparent homologue of cor15a, is strongly responsive to cold and ABA, but not drought. Plant Molecular Biology, 1993, 23(5): 1073–1077 https://doi.org/10.1007/BF00021822 pmid: 8260628
30	Y Cao, X Xiang, M Geng, Q You, X Huang. Effect of HbDHN1 and HbDHN2 genes on abiotic stress responses in Arabidopsis. Frontiers of Plant Science, 2017, 8: 470 https://doi.org/10.3389/fpls.2017.00470 pmid: 28443102
31	A L Saucedo, E E Hernández-Domínguez, L A de Luna-Valdez, A A Guevara-García, A Escobedo-Moratilla, E Bojorquéz-Velázquez, F Del Río-Portilla, D A Fernández-Velasco, A P Barba de la Rosa. Insights on structure and function of a late embryogenesis abundant protein from Amaranthus cruentus: an intrinsically disordered protein involved in protection against desiccation, oxidant conditions, and osmotic stress. Frontiers of Plant Science, 2017, 8: 497 https://doi.org/10.3389/fpls.2017.00497 pmid: 28439280
32	S B Mowla, A Cuypers, S P Driscoll, G Kiddle, J Thomson, C H Foyer, F L Theodoulou. Yeast complementation reveals a role for an Arabidopsis thaliana late embryogenesis abundant (LEA)-like protein in oxidative stress tolerance. Plant Journal, 2006, 48(5): 743–756 https://doi.org/10.1111/j.1365-313X.2006.02911.x pmid: 17092320
33	F Bao, D Du, Y An, W Yang, J Wang, T Cheng, Q Zhang. Overexpression of Prunus mume dehydrin genes in tobacco enhances tolerance to cold and drought. Frontiers of Plant Science, 2017, 8: 151 https://doi.org/10.3389/fpls.2017.00151 pmid: 28224001
34	B Saha, S Mishra, J P Awasthi, L Sahoo, S K Panda. Enhanced drought and salinity tolerance in transgenic mustard [Brassica juncea (L.) Czern & Coss.] overexpressing Arabidopsis group 4 late embryogenesis abundant gene (AtLEA4–1). Environmental and Experimental Botany, 2016, 128: 99–111 https://doi.org/10.1016/j.envexpbot.2016.04.010
35	J Yu, Y Lai, X Wu, G Wu, C Guo. Overexpression of OsEm1 encoding a group I LEA protein confers enhanced drought tolerance in rice. Biochemical and Biophysical Research Communications, 2016, 478(2): 703–709 https://doi.org/10.1016/j.bbrc.2016.08.010 pmid: 27524243

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