Overexpression of Cassava (Manihot esculenta) ABA Receptor MePYL4a in Arabidopsis thaliana Enhances Its Drought Tolerance
ZHAO Dong-Li1,2, BAO Ru-Xue1,2, LI Meng-Tao1,2, CHEN Xin2,*, WANG Wen-Quan1,*
1 College of Tropical Crops, Hainan University, Haikou 570228, China; 2 Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Abstract：Abscisic acid (ABA) and its receptor pyrabactin resistance like (PYL) play an important role in plant resistance to drought stress. In order to explore the drought resistance function of the MePYL4a (GenBank No. XM_021766535) gene of cassava (Manihot esculenta), the MePYL4a gene was cloned from the cassava variety 'SC8', and its bioinformatics analysis was carried out. The subcellular localization was carried out using the Agrobacterium tumefaciens mediated transient transformation method; The expression of MePYL4a gene under drought stress was detected by qPCR; MePYL4a overexpression vector was constructed and transformed into Arabidopsis thaliana for drought resistance analysis of A. thaliana transgenic plants. The results showed that MePYL4a was an unstable protein, and the subcellular localization characteristics of MePYL4a protein showed that it was located in the nucleus and cytoplasm; qPCR analysis showed that MePYL4a gene expression level was significantly induced by drought stress, reached the highest level in 1 h, and then slowly decreased. After drought treatment, the phenotype observation showed that MePYL4a could improve the drought tolerance of A. thaliana seedlings under drought stress. The average biomass of over expressed plants was 1.5 times that of wide type (WT). The expression of key rate limiting enzyme gene 9-cis-epoxycarotenoid dioxygenase (NCED3), and drought stress response index gene responsive to dehydration (RD29A) for ABA synthesis was significantly higher than that of WT and Atpyl4 mutant plant. And the activity of antioxidant enzymes was about twice higher than that of WT, indicating that the ability of over expressed plants of MePYL4a to remove active oxygen was stronger than that of WT and Atpyl4 plant. Under drought stress, it could accumulate more biomass and enhance the drought resistance of A. thaliana. In conclusion, MePYL4a overexpression reduced the accumulation of reactive oxygen species in A. thaliana under drought stress and induced higher expression of stress resistance genes, which enhanced drought resistance of A. thaliana. This study provides a reference for MePYL4a to participate in cassava stress resistance.
[1] 颜彦, 铁韦韦, 丁泽红, 等. 2018. 木薯MePYL8基因克隆及表达分析[J]. 分子植物育种, 16(14): 4498-4504. (Yan Y, Tie W W, Ding Z H, et al.2018. Cloning and expression analysis of MePYL8 gene in cassava[J]. Molecular Plant Breeding, 16(14): 4498-4504.) [2] 郑清岭, 杨冬艳, 刘建文, 等. 2017. 干旱胁迫对沙芥和斧形沙芥幼苗生长及抗氧化系统的影响[J]. 植物生理学报, 53(4): 600-608. (Zheng Q L, Yang D Y, Liu J W, et al.2017. Effects of drought stress on the growth and antioxidant system of sand mustard and axe sand mustard seedlings[J]. Chinese Journal of Plant, 53(4): 600-608.) [3] Asha A D, Nivetha N, Krishna G K, et al.2021. Amelioration of short-term drought stress during different growth stages in Brassica juncea by rhizobacteria mediated maintenance of ROS homeostasis[J]. Physiologia Plantarum, 172(4): 1880-1893. [4] Assmann S M, Jegla T.2016. Guard cell sensory systems: Recent insights on stomatal responses to light, abscisic acid, and CO2[J]. Current Opinion in Plant Biology, 33:157-167. [5] Bai G, Xie H, Yao H, et al.2019. Genome-wide identification and characterization of ABA receptor PYL/RCAR gene family reveals evolution and roles in drought stress in Nicotiana tabacum[J]. BMC Genomics, 20(1): 575-593. [6] Boscolo A, Starr J, Sanchez V, et al.2012. The abolishment of anesthesia-induced cognitive impairment by timely protection of mitochondria in the developing rat brain: The importance of free oxygen radicals and mitochondrial integrity[J]. Neurobiology of Disease, 45(3): 1031-1041. [7] Chater C, Peng K, Movahedi M, et al.2015. Elevated CO2-induced responses in stomata require ABA and ABA signaling[J]. Current Biology, 25(20): 2709-2716. [8] Dittrich M, Mueller H M, Bauer H, et al.2019. The role of Arabidopsis ABA receptors from the PYR/PYL/RCAR family in stomatal acclimation and closure signal integration[J]. Nature Plants, 5(9): 1002-1011. [9] Fujii H, Chinnusamy V, Rodrigues A, et al.2009. In vitro reconstitution of an abscisic acid signalling pathway[J]. Nature, 462(7273): 660-664. [10] Ge Y, He X, Wang J, et al.2014. Physiological and biochemical responses of phoebe bournei seedlings to water stress and recovery[J]. Acta Physiologiae Plantarum, 36(5): 1241-1250. [11] Habib S, Lwin Y Y, Li N.2021. Down-regulation of SlGRAS10 in tomato confers abiotic stress tolerance[J]. Genes (Basel), 12(5): 623-643. [12] He Z, Zhong J, Sun X, et al.2018.The maize ABA receptors ZmPYL8, 9, and 12 facilitate plant drought resistance[J]. Frontiers in Plant Science, 9: 422-433. [13] Hu X, Zhang A, Zhang J, Jiang M, et al.2006. Abscisic acid is a key inducer of hydrogen peroxide production in leaves of maize plants exposed to water stress[J]. Plant Cell Physiology, 47(11): 1484-1495. [14] Iuchi S, Kobayashi M, Taji T, et al.2001. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis[J]. Pant Journal, 27(4): 325-333. [15] Jakab G, Ton J, Flors V, et al.2005. Enhancing Arabidopsis salt and drought stress tolerance by chemical priming for its abscisic acid responses[J]. Plant Physiology, 139(1): 267-274. [16] Kim H, Hwang H, Hong J W, et al.2012. A rice orthologue of the ABA receptor, OsPYL/RCAR5, is a positive regulator of the ABA signal transduction pathway in seed germination and early seedling growth[J]. Journal of Experimental Botany, 63(2): 1013-1024. [17] Kim H, Lee K, Hwang H, et al.2014. Overexpression of PYL5 in rice enhances drought tolerance, inhibits growth, and modulates gene expression[J]. Journal of Experimental Botany, 65(2): 453-464. [18] Krasensky J, Jonak C.2012. Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks[J]. Journal of Experimental Botany, 63(4):1593-1608. [19] Lawlor D W, Tezara W.2009. Causes of decreased photosynthetic rate and metabolic capacity in water-deficient leaf cells: A critical evaluation of mechanisms and integration of processes[J]. Annals of Botany, 103(4): 561-579. [20] Lee S C, Luan S.2012. ABA signal transduction at the crossroad of biotic and abiotic stress responses[I]. Plant Cell Environment, 35(1): 53-60. [21] Li T, Wang R, Zhao D, et al.2020. Effects of drought stress on physiological responses and gene expression changes in herbaceous peony (Paeonia lactiflora Pall.)[J]. Plant Signaling & Behavior, 15(5): 1746034. [22] Liang C, Liu Y, Li Y, et al.2017. Activation of ABA receptors gene GhPYL9-11A is positively correlated with cotton drought tolerance in transgenic Arabidopsis[J]. Frontiers in Plant Science, 8: 1453-1465. [23] Luo X, Li C, He X, et al.2020. ABA signaling is negatively regulated by GbWRKY1 through JAZ1 and ABI1 to affect salt and drought tolerance[J]. Plant Cell Reports, 39(2): 181-194. [24] Ma Y, Szostkiewicz I, Korte A, et al.2009. Regulators of PP2C phosphatase activity function as abscisic acid sensors[J]. Science, 324(5930): 1064-1068. [25] Mahmood T, Khalid S, Abdullah M, et al.2019. Insights into drought stress signaling in plants and the molecular genetic basis of cotton drought tolerance[J]. Cells, 319(1):105-134. [26] Mega R, Abe F, Kim J S, et al.2019. Tuning water-use efficiency and drought tolerance in wheat using abscisic acid receptors[J]. Nature Plants, 5(2): 153-159. [27] Modarresi M, Eyvazi A.2014. Bioinformatical evaluation of desiccation-responsive rd29A gene in Arabidopsis thaliana[J]. Pakistan Journal of Biological Sciences, 17(1): 80-85. [28] Nambara E, Marion-Poll A.2005. Abscisic acid biosynthesis and catabolism[J]. Annual Review of Plant Biology, 56(1): 165-185. [29] Nai G, Liang G, Ma W,et al.2022. Overexpression VaPYL9 improves cold tolerance in tomato by regulating key genes in hormone signaling and antioxidant enzyme[J].BMC Plant Biology, 22(1): 344-361. [30] Nilson S E, Assmann S M.2007. The control of transpiration. insights from Arabidopsis[J]. Plant Physiology. 143(1): 19-27. [31] Park S Y, Fung P, Nishimura N, et al.2009. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins[J]. Science, 324(5930): 1068-1071. [32] Pizzio G A, Rodriguez L, Antoni R, et al.2013. The PYL4 A194T mutant uncovers a key role of PYR1-LIKE4/PROTEIN PHOSPHATASE 2CA interaction for abscisic acid signaling and plant drought resistance[J]. Plant Physiology, 163(1): 441-455. [33] Qin Y X, Wang M C, Tian Y C, et al.2012. Over-expression of TaMYB33 encoding a novel wheat MYB transcription factor increases salt and drought tolerance in Arabidopsis[J]. Molecular Biology Reports, 39(6): 7183-7192. [34] Reddy A R, Chaitanya K V, Vivekanandan M.2004. Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants[J]. Journal of Plant Physiology, 161(11): 1189-1202. [35] Verma R K, Santosh K V V, Yadav S K, et al.2019. Overexpression of ABA receptor PYL10 gene confers drought and cold tolerance to Indica rice[J]. Frontiers in Plant Science, 10: 1488-1503. [36] Wang L Q, Li Z, Wen S S, et al.2020. WUSCHEL-related homeobox gene PagWOX11/12a responds to drought stress by enhancing root elongation and biomass growth in poplar[J]. Journal of Experimental Botany 71(4):1503-1513. [37] Xie H, Yang D H, Yao H, et al.2016. ITRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress[J]. Biochemical and Biophysical Research Communications, 469(3): 768-775. [38] Yang Z, Liu J, Poree F, et al.2019. Abscisic acid receptors and coreceptors modulate plant water use efficiency and water productivity[J]. Plant Physiology, 180(2): 1066-1080. [39] Yu J, Ge H, Wang X, et al.2017. Overexpression of pyrabactin resistance-like abscisic acid receptors enhances drought, osmotic, and cold tolerance in transgenic poplars[J]. Frontiers in Plant Science, 8: 1752-1764. [40] Zhang A, Liu M, Gu W, et al.2021. Effect of drought on photosynthesis, total antioxidant capacity, bioactive component accumulation, and the transcriptome of Atractylodes lancea[J]. BMC Plant Biology, 21(1): 293-306. [41] Zhao Y, Chan Z, Gao J, et al.2016. ABA receptor PYL9 promotes drought resistance and leaf senescence[J]. Proceedings of the National Academy of Sciences of the USA, 113(7): 1949-1954. [42] Ziska L, Runion G, Tomecek M, et al.2009. An evaluation of cassava, sweet potato and field corn as potential carbohydrate sources for bio-ethanol production in Alabama and Maryland[J]. Biomass and Bioenergy, 33(11): 1503-1508.
