Abstract
External application of ethanol enhances tolerance to high salinity, drought, and heat stress in various plant species. However, the effects of ethanol application on increased drought tolerance in woody plants, such as the tropical crop “cassava,” remain unknown. In the present study, we analyzed the morphological, physiological, and molecular responses of cassava plants subjected to ethanol pretreatment and subsequent drought stress treatment. Ethanol pretreatment induced a slight accumulation of abscisic acid (ABA) and stomatal closure, resulting in a reduced transpiration rate, higher water content in the leaves during drought stress treatment and the starch accumulation in leaves. Transcriptomic analysis revealed that ethanol pretreatment upregulated the expression of ABA signaling-related genes, such as PP2Cs and AITRs, and stress response and protein-folding-related genes, such as heat shock proteins (HSPs). In addition, the upregulation of drought-inducible genes during drought treatment was delayed in ethanol-pretreated plants compared with that in water-pretreated control plants. These results suggest that ethanol pretreatment induces stomatal closure through activation of the ABA signaling pathway, protein folding-related response by activating the HSP/chaperone network and the changes in sugar and starch metabolism, resulting in increased drought avoidance in plants.
Key message
Ethanol priming induces drought stress avoidance in cassava by regulating stomatal closure.
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The microarray data were deposited in the GEO database (GSE195521). https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE195521. The secure token for the reviewers: uxshyocedrofxqn.
References
Aina OO, Dixon AGO, Akirinde EA (2007) Effect of soil moisture stress on growth and yield of cassava in Nigeria. Pakistan J Bio Sci 10(18):3085–3090
Bashir K, Todaka D, Rasheed S, Matsui A, Ahmad Z, Sako K, Utsumi Y, Vu AT, Tanaka M, Takahashi S, Ishida J, Tsuboi Y, Watanabe S, Kanno Y, Ando E, Shin KC, Seito M, Motegi H, Sato M, Li R, Kikuchi S, Fujita M, Kusano M, Kobayashi M, Habu Y, Nagano AJ, Kawaura K, Kikuchi J, Saito K, Hirai MY, Seo M, Shinozaki K, Kinoshita T, Seki M (2022) Ethanol-mediated novel survival strategy against drought stress in plants. Plant Cell Physiol (in press). https://doi.org/10.1093/pcp/pcac114
Bhaskara GB, Nguyen TT, Verslues PE (2012) Unique drought resistance functions of the highly ABA-induced clade a protein phosphatase 2Cs. Plant Physiol 160:379–395
Chemonges M, Balyyeiusa EK, Bisikwa J, Orisu DSO (2013) Phenotypic and physiological traits associated with drought tolerant cassava cultivars in Uganda. Afr Crop Conf Proceed 11:463–469
Clement M, Leonhardt N, Droillard MJ, Reiter I, Montillet JL, Genty B, Lauriere C, Nussaume L, Noel LD (2011) The cytosolic/nuclear HSC70 and HSP90 molecular chaperones are important for stomatal closure and modulate abscisic acid-dependent physiological responses in Arabidopsis. Plant Physiol 156:1481–1492
EL-Sharkary MA (2004) Cassava biology and physiology. Plant Mol Biol 56:481–501
El-Sharkawy MA (2007) Physiological characteristic of cassava tolerance to prolonged drought in the tropics: implications for breeding cultivars adapted to seasonally dry and semiarid environments. Braz J Plant Physiol 19:257–286
Fu L, Ding Z, Han B, Hu W, Li Y, Zhang J (2016) Physiological investigation and transcriptome analysis of polyethylene glycol (PEG)-induced dehydration stress in cassava. Int J Mol Sci 17:283
Guttikonda SK, Valliyodan B, Neelakandan AK, Tran LSP, Kumar R, Quach TN, Voothuluru P, Gutierrez-Gonzalez JJ, Aldrich DL, Pallardy SG, Sharp RE, Ho THD, Nguyen HT (2014) Overexpression of AtDREB1D transcription factor improves drought tolerance in soybean. Mol Biol Rep 41:7995–8008
Ha CV, Leyva-Gonzalez MA, Osakabe Y, Tran UT, Nishiyama R, Watanabe Y, Tanaka M, Seki M, Yamaguchi S, Dong NV, Yamaguchi-Shinozaki K, Shinozaki K, Herrera-Estrella L, Tran LS (2014) Positive regulatory role of strigolactone in plant responses to drought and salt stress. Proc Natl Acad Sci U S A 111:851–856
Hu M, Hu W, Xia Z, Zhou X, Wang W (2016) Validation of reference genes for relative quantitative gene expression studies in cassava (Manihot esculenta Crantz) by using quantitative real-time PCR. Front Plant Sci 7:680
Jacob P, Hirt H, Bendahmane A (2017) The heat-shock protein/chaperone network and multiple stress resistance. Plant Biotechnol J 15:405–414
Jolayemi OL, Opabode JT (2018) Response of cassava (Manihot esculenta Crantz) varieties to in vitro mannitol-induced drought stress. J Crop Improv 32:566–578
Kanno Y, Oikawa T, Chiba Y, Ishimaru Y, Simizu T, Sano N (2016) AtSWEET13 and AtSWEET14 regrulate gibberellin-mediated physilogical processes. Nat Commun 7:13245
Khan MI, Fatma M, Per TS, Anjum NA, Khan NA (2015) Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants. Front Plant Sci 6:462
Kim JM, To TK, Matsui A, Tanoi K, Kobayashi NI, Matsuda F, Habu Y, Ogawa D, Sakamoto T, Matsunaga S, Bashir K, Rasheed S, Ando M, Takeda H, Kawaura K, Kusano M, Fukushima A, Endo TA, Kuromori T, Ishida J, Morosawa T, Tanaka M, Torii C, Takebayashi Y, Sakakibara H, Ogihara Y, Saito K, Shinozaki K, Devoto A, Seki M (2017) Acetate-mediated novel survival strategy against drought in plants. Nat Plants 3:17097
Kinoshita T, Seki M (2014) Epigenetic memory for stress response and adaptation in plants. Plant Cell Physiol 55:1859–1863
Krishna P, Gloor G (2001) The Hsp90 family of proteins in Arabidopsis thaliana. Cell Stress Chaperones 6:238–246
Kumar D, Yusuf MA, Singh P, Sardar M, Sarin NB (2014) Histochemical detection of superoxide and H2O2 accumulation in Brassica juncea seedlings. Bio Protoc 4:e1108
Leng L, Liang Q, Jiang J, Zhang C, Hao Y, Wang X, Su W (2017) A subclass of HSP70s regulate development and abiotic stress responses in Arabidopsis thaliana. J Plant Res 130:349–363
Li G, Li J, Hao R, Guo Y (2017) Activation of catalase activity by a peroxisome-localized small heat shock protein Hsp17.6CII. J Genet Genom 44:395–404
Lobell DB, Schlenker W, Costa-Roberts J (2011) Climate trends and global crop production since 1980. Science 333:616–620
Ma Y, Tian H, Lin R, Wang W, Zhang N, Hussain S, Yang W, Zhang C, Zhou G, Wang T, Wang S (2021) AITRL, an evolutionarily conserved plant specific transcription repressor regulates ABA response in Arabidopsis. Sci Rep 11:721
Malik AI, Kongsil P, Nguyễn VA, Ou W, Sholihin, Srean P, Sheela MN, Lopez-Lavalle LAB, Utsumi Y, Lu C, Kittipadakul P, Nguyễn HH, Ceballos H, Nguyễn TH, Gomez MS, Aiemnaka P, Labarta R, Chen S, Amawan S, Sok S, Youabee L, Seki M, Tokunaga H, Wang W, Li K, Nguyễn HA, Nguyễn VD, Hàm LH, Ishitani M (2020) Cassava breeding and agronomy in Asia: 50 years of history and future directions. Breed Sci 70:145–166
Maruri-Lopez I, Figueroa NE, Hernandez-Sanchez IE, Chodasiewicz M (2021) Plant stress granules: trends and beyond. Front Plant Sci 12:722643
Matsui A, Todaka D, Tanaka M, Mizunashi K, Takahashi S, Sunaoshi Y, Tsuboi Y, Ishida J, Bashir K, Kikuchi J, Kusano M, Kobayashi M, Kawaura K, Seki M (2022) Ethanol induces heat tolerance in plants by stimulating unfolded protein response. Plant Mol Biol (in press). https://doi.org/10.1007/s11103-022-01291-8
Mostofa MG, Rahman A, Ansary MM, Watanabe A, Fujita M, Tran LS (2015) Hydrogen sulfide modulates cadmium-induced physiological and biochemical responses to alleviate cadmium toxicity in rice. Sci Rep 5:14078
Muthusamy SK, Dalal M, Chinnusamy V, Bansal KC (2017) Genome-wide identification and analysis of biotic and abiotic stress regulation of small heat shock protein (HSP20) family genes in bread wheat. J Plant Physiol 211:100–113
Naullage PM, Haghighatlari M, Namini A, Teixeira JMC, Li J, Zhang O, Gradinaru CC, Forman-Kay JD, Head-Gordon T (2022) Protein dynamics to define and refine disordered protein ensembles. J Phys Chem B 126:1885–1894
Necci M, Piovesan D, Dosztanyi Z, Tosatto SCE (2017) MobiDB-lite: Fast and highly specific consensus prediction of intrinsic disorder in proteins. Bioinformatics 33:1402–1404
Nguyen HM, Sako K, Matsui A, Suzuki Y, Mostofa MG, Ha CV, Tanaka M, Tran LP, Habu Y, Seki M (2017) Ethanol enhances high-salinity stress tolerance by detoxifying reactive oxygen species in Arabidopsis thaliana and Rice. Front Plant Sci 8:1001
Nguyen HM, Sako K, Matsui A, Ueda M, Tanaka M, Ito A, Nishino N, Yoshida M, Seki M (2018) Transcriptomic analysis of Arabidopsis thaliana plants treated with the Ky-9 and Ky-72 histone deacetylase inhibitors. Plant Signal Behav 13:e1448333
Nishiyama R, Watanabe Y, Fujita Y, Le DT, Kojima M, Werner T, Vankova R, Yamaguchi-Shinozaki K, Shinozaki K, Kakimoto T, Sakakibara H, Schmulling T, Tran LS (2011) Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis. Plant Cell 23:2169–2183
Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, Craggs TD, Bazett-Jones DP, Pawson T, Forman-Kay JD, Baldwin AJ (2015) Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol Cell 57:936–947
Oliveira EJD, Aidar SDT, Morgate CV, Chaves ARDM, Cruz JL, Coelho Filho MA (2015) Genetic parameters for drought-tolerance in cassava. Pesq Agropec Bras 50:233–241
Okogbenin E, Setter TL, Ferguson M, Mutegi R, Ceballos H, Olasanmi B, Fregene M (2013) Phenotypic approaches to drought in cassava: review. Front Physiol 4:93
Orek C, Gruissem W, Ferguson M, Vanderschuren H (2020) Morpho-physiological and molecular evaluation of drought tolerance in cassava (Manihot esculenta Crantz). Field Crops Res 255:107861
Patanun O, Ueda M, Itouga M, Kato Y, Utsumi Y, Matsui A, Tanaka M, Utsumi C, Sakakibara H, Yoshida M, Narangajavana J, Seki M (2016) The histone deacetylase inhibitor suberoylanilide hydroxamic acid alleviates salinity stress in cassava. Front Plant Sci 7:2039
Rasheed S, Bashir K, Kim JM, Ando M, Tanaka M, Seki M (2018) The modulation of acetic acid pathway genes in Arabidopsis improves survival under drought stress. Sci Rep 8:7831
Ren MY, Feng RJ, Shi HR, Lu LF, Yun TY, Peng M, Guan X, Zhang H, Wang JY, Zhang XY, Li CL, Chen YJ, He P, Zhang YD, Xie JH (2017) Expression patterns of members of the ethylene signaling–related gene families in response to dehydration stresses in cassava. PLoS ONE 12:e0177621
Rosenthal DM, Ort DR (2012) Examining cassava’s potential to enhance food security under climate change. Tropical Plant Biol 5:30–38
Sako K, Kim JM, Matsui A, Nakamura K, Tanaka M, Kobayashi M, Saito K, Nishino N, Kusano M, Taji T, Yoshida M, Seki M (2016) Ky-2, a histone deacetylase inhibitor, enhances high-salinity stress tolerance in Arabidopsis thaliana. Plant Cell Physiol 57:776–783
Sako K, Nguyen HM, Seki M (2020) Advances in chemical priming to enhance abiotic stress tolerance in Plants. Plant Cell Physiol 61:1995–2003
Sako K, Nagashima R, Tamoi M, Seki M (2021) Exogenous ethanol treatment alleviates oxidative damage of Arabidopsis thaliana under conditions of high-light stress. Plant Biotechnol (tokyo) 38:339–344
Sakamoto H, Maruyama K, Sakuma Y, Meshi T, Iwabuchi M, Shinozaki K (2004) Yamaguchi-Shinozaki K (2004) Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiol 136:2734–2746
Savvides A, Ali S, Tester M, Fotopoulos V (2016) Chemical priming of plants against multiple abiotic stresses: mission possible? Trends Plant Sci 21:329–340
Shin Y, Brangwynne CP (2017) Liquid phase condensation in cell physiology and disease. Science 357:eaaf4382
Stein O, Granot D (2019) An overview of sucrose synthases in plants. Front Plant Sci 10:95
Smith SM, Fulton DC, Chia T, Thorneycroft D, Chapple A, Dunstan H, Hylton C, Zeeman SC, Smith AM (2004) Diurnal changes in the transcriptome encoding enzymes of starch metabolism provide evidence for both transcriptional and posttranscriptional regulation of starch metabolism in Arabidopsis leaves. Plant Physiol 136:2687–2699
Tadege M, Dupuis II, Kuhlemeier C (1999) Ethanolic fermentation: new functions for an old pathway. Trends Plant Sci 4:320–325
Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K (2002) Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. Plant J 29:417–426
Tang T, Yu A, Li P, Yang H, Liu G, Liu L (2016) Sequence analysis of the Hsp70 family in moss and evaluation of their functions in abiotic stress responses. Sci Rep 6:33650
Todaka D, Zhao Y, Yoshida T, Kudo M, Kidokoro S, Mizoi J, Kodaira KS, Takebayashi Y, Kojima M, Sakakibara H, Toyooka K, Sato M, Fernie AR, Shinozaki K, Yamaguchi-Shinozaki K (2017) Temporal and spatial changes in gene expression, metabolite accumulation and phytohormone content in rice seedlings grown under drought stress conditions. Plant J 90:61–78
Turyagyenda LF, Kizito EB, Ferguson M, Baguma Y, Agaba M, Harvey JJ, Osiru DS (2013) Physiological and molecular characterization of drought responses and identification of candidate tolerance genes in cassava. AoB Plants 5:plt007
Uddling J, Gelang-Alfredsson J, Piikki K, Pleijel H (2007) Evaluating the relationship between leaf chlorophyll concentration and SPAD-502 chlorophyll meter readings. Photosyn Res 91:37–46
Ueda M, Matsui A, Tanaka M, Nakamura T, Abe T, Sako K, Sasaki T, Kim JM, Ito A, Nishino N, Shimada H, Yoshida M, Seki M (2017) The distinct roles of class I and II RPD3-like histone deacetylases in salinity stress response. Plant Physiol 175:1760–1773
Utsumi Y, Utsumi C, Sawada T, Fujita N, Nakamura Y (2011) Functional diversity of isoamylase oligomers: The ISA1 homo-oligomer is essential for amylopectin biosynthesis in rice endosperm. Plant Physiol 156:61–77
Utsumi Y, Tanaka M, Morosawa T, Kurotani A, Yoshida T, Mochida K, Matsui A, Umemura Y, Ishitani M, Shinozaki K, Sakurai T, Seki M (2012) Transcriptome analysis using a high-density oligomicroarray under drought stress in various genotypes of cassava: an important tropical crop. DNA Res 19:335–345
Utsumi Y, Tanaka M, Kurotani A, Yoshida T, Mochida K, Matsui A, Ishitani M, Sraphet S, Whankaew S, Asvarak T, Narangajavana J, Triwitayakorn K, Sakurai T, Seki M (2016) Cassava (Manihot esculenta) transcriptome analysis in response to infection by the fungus Colletotrichum gloeosporioides using an oligonucleotide-DNA microarray. J Plant Res 129:711–726
Utsumi Y, Utsumi C, Tanaka M, Ha VT, Matsui A, Takahashi S, Seki M (2017) Formation of friable embryogenic callus in cassava is enhanced under conditions of reduced nitrate, potassium and phosphate. PLoS ONE 12:e0180736
Utsumi Y, Utsumi C, Tanaka M, Ha CV, Takahashi S, Matsui A, Matsunaga TM, Matsunaga S, Kanno Y, Seo M, Okamoto Y, Moriya E, Seki M (2019) Acetic acid treatment enhances drought avoidance in Cassava (Manihot esculenta Crantz). Front Plant Sci 10:521
Varshney RK, Tuberosa R, Tardieu F (2018) Progress in understanding drought tolerance: from alleles to cropping systems. J Exp Bot 69:3175–3179
Wu TY, Juan YT, Hsu YH, Wu SH, Liao HT, Fung RWM, Charng YY (2013) Interplay between heat shock proteins HSP101 and HSA32 prolongs heat acclimation memory posttranscriptionally in Arabidopsis. Plant Physiol 161:2075–2084
Yoon JY, Hamayun M, Lee S-K, Lee I-J (2009) Methyl jasmonate alleviated salinity stress in soybean. J Crop Sci Biotechnol 12:63–68
Zhao P, Liu P, Shao J, Li C, Wang B, Guo X, Yan B, Xia Y, Peng M (2015) Analysis of different strategies adapted by two cassava cultivars in response to drought stress: ensuring survival or continuing growth. J Exp Bot 66:1477–1488
Acknowledgements
We would like to thank Editage (www.editage.com) for English language editing.
Funding
This work was supported by grants from RIKEN, Japan (to MS), including the RIKEN–AIST Joint Research Fund (full research), Core Research for Evolutionary Science and Technology (JPMJCR13B4 to MS), and A-STEP (JPMJTM19BS to MS) of the Japan Science and Technology Agency (JST), and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (Innovative Areas 18H04791 and 18H04705 to MS).
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YU and MoS designed the study. ATV, YU, CU, and MT measured the physiological parameters. YK and MiS performed phytohormone measurements. ATV, YU, and MT prepared RNA and performed microarray analysis. DA and ST performed the prediction of intrinsically disordered regions and helped with transcriptome analysis. YU, ATV, and CU propagated the plants. EA and TK measured stomatal apertures. KO,KB and XHP provided comments. YU, ATV, and MoS wrote the original draft.
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11103_2022_1300_MOESM1_ESM.tif
Supplementary Figure 1. DAB staining of cassava leaves. a DAB staining of the leaves from plants pretreated with 1.0% ethanol or water (control) for 5 days. b DAB staining of the leaves from cassava plants pretreated with 1.0% ethanol and water (control) and then subjected to drought stress treatments for 6 days. c DAB staining of the leaves from cassava plants pretreated with 1.0% ethanol and water (control) and then subjected to drought stress treatment for 12 days
11103_2022_1300_MOESM2_ESM.tif
Supplementary Figure 2. Representative cassava plants that were pretreated with 1% ethanol and water (control) and then subjected to drought stress treatment for 6 days. Bar = 30 cm
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Vu, A.T., Utsumi, Y., Utsumi, C. et al. Ethanol treatment enhances drought stress avoidance in cassava (Manihot esculenta Crantz). Plant Mol Biol 110, 269–285 (2022). https://doi.org/10.1007/s11103-022-01300-w
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DOI: https://doi.org/10.1007/s11103-022-01300-w