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Ethanol treatment enhances drought stress avoidance in cassava (Manihot esculenta Crantz)

August 2022
Plant Molecular Biology 110(18)

Authors:

RIKEN

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.

Analysis of leaf wilting, leaf water content, and chlorophyll content in ethanol- and water-pretreated cassava plants during drought stress. a Representative cassava plants that were pretreated with 1% ethanol and water (control) and then subjected to drought stress treatment for 12 days. Bar = 10 cm. b Relative water content of the leaves of cassava plants 6 days after the commencement of soil drying. Means and SD from measurements of 6 biological replicates are shown

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Analysis of chlorophyll content in ethanol- and water-pretreated cassava plants during drought stress. a Relative content of water remaining on the pot with 1.0% ethanol-pretreated- (orange line) and water-pretreated (blue line) plants after the exposure to drought stress. One asterisk indicates a significant difference (p ≤ 0.05) between the results from ethanol pretreatment and those from water pretreatment on the same day after the commencement of soil drying. Means and SD from measurements of 30 biological replicates are shown. p-value was estimated using Student’s t-test. b SPAD levels in the leaves of 1.0% ethanol-pretreated (orange bar) and water-pretreated (blue bar) cassava plants after 6 days of commencement of soil drying. Means and SD from measurements of 6 biological replicates are shown. One asterisk indicates a difference (*p ≤ 0.05) between the SPAD value from ethanol pretreatment. P-value was calculated using Student’s t-test. p-value in leaf positions 8 and 9 was not calculated due to the dropping of the leaves. c Number of fine, yellow, and withered cassava leaves in the plants pretreated with 1.0% ethanol (orange bar) and water (blue bar) on days 4 and 6 after the commencement of soil drying. Means and SD from measurements of 12 biological replicates are shown. One asterisk indicates a significant difference (*p ≤ 0.05) between the results of two subsequent measurements based on Student’s t-test

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Effects of ethanol pretreatment on stomatal closure, transpiration rate, and photosynthetic rate in cassava plants. a Thermal images of cassava plants pretreated with either ethanol or water for 5 days. b Net photosynthetic rate, stomatal conductance, and transpiration rate for the first to third leaves below the stem apices of cassava plants pretreated with ethanol or water (control) for 5–6 days. Bars with the same letter indicate that they are not significantly different (p ≤ 0.05). The p-value was calculated using a one-way ANOVA with Turkey-HSD significant difference multiple comparisons test. Mean and SD were measured from 6 biological replicates. c Stomatal aperture of cassava plants pretreated with either ethanol or water for 5–6 days. Mean and SD were measured from 2 biological replicates. P-value was calculated using Student’s t-test

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Effects of ethanol pretreatment on the ABA, JA, and JA-Ile contents of cassava leaves. E5, W5, E5D6, W5D6, E5D12, and W5D12 represent the following: leaf samples from plants pretreated with 1.0% ethanol for 5 days (E5), samples pretreated with water for 5 days (W5), samples from the plants exposed to drought stress for 6 days following pretreatment with 1.0% ethanol for 5 days (E5D6), samples from the plants exposed to drought stress for 6 days following pretreatment with water for 5 days (W5D6), samples from plants exposed to drought stress for 12 days following pretreatment with 1.0% ethanol for 5 days (E5D12), and samples from plants exposed to drought stress for 12 days following pretreatment with water for 5 days (W5D12). Mean ± SE represents the data of four biological replicates. p-value on the figure was measured using Student’s t-test

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Hypothetical model of ethanol-mediated increased drought avoidance in cassava. Our data suggest that ethanol pretreatment upregulates the following three pathways before the drought stress treatment: (1) Induction of stomatal closure by upregulation of the ABA signaling network; (2) Regulation of protein folding by activation of the HSP chaperon network; (3) Increase of the accumulation of transitory starch. After the drought stress treatment, upregulation of drought-inducible gene expression is delayed and the expression of some drought stress tolerance-related genes is upregulated. Then, the plants maintain water content under drought stress, which increases drought stress avoidance

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Vol.:(0123456789)

1 3

Plant Molecular Biology (2022) 110:269–285

https://doi.org/10.1007/s11103-022-01300-w

Ethanol treatment enhances drought stress avoidance incassava

(Manihot esculenta Crantz)

AnhThuVu1,7· YoshinoriUtsumi1 · ChikakoUtsumi1· MahoTanaka1,2· SatoshiTakahashi1,2· DaisukeTodaka1·

YuriKanno3· MitsunoriSeo3· EigoAndo4· KaoriSako1,5· KhurramBashir1,6· ToshinoriKinoshita7· XuanHoiPham8·

MotoakiSeki1,2,9

Received: 14 March 2022 / Accepted: 13 July 2022 / Published online: 15 August 2022

Abstract

External application of ethanol enhances tolerance to high salinity, drought, and heat stress in various plant species. How-

ever, 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 accu-

mulation 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.

Keywords Cassava· Drought avoidance· Ethanol· Stomatal closure· Sugar and starch metabolism

Anh Thu Vu and Yoshinori Utsumi have contributed equally to this

work.

* Yoshinori Utsumi

yoshinori.utsumi@riken.jp

* Motoaki Seki

motoaki.seki@riken.jp

1 Plant Genomic Network Research Team, RIKEN Center

forSustainable Resource Science (CSRS), 1-7-22

Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa230-0045,

Japan

2 Plant Epigenome Regulation Laboratory, RIKEN

Cluster forPioneering Research, 2-1 Hirosawa, Wako,

Saitama351-0198, Japan

3 Dormancy andAdaptation Research Unit, RIKEN Center

forSustainable Resource Science, Yokohama, Japan

4 Department ofBiological Sciences, School ofScience,

The University ofTokyo, 7-3-1 Hongo, Bunkyo,

Tokyo113-0033, Japan

5 Department ofAdvanced Bioscience, Faculty ofAgriculture,

Kindai University, Nara631-8505, Japan

6 Department ofLife Sciences, Lahore University

ofManagement Sciences, Lahore, Pakistan

7 Institute ofTransformative Bio-Molecules (WPI-ITbM),

Nagoya University, Chikusa, Nagoya464-8602, Japan

8 Agricultural Genetics Institute, Pham Van Dong Road, Bac

Tu Lie District, HaNoi, Vietnam

9 Kihara Institute forBiological Research, Yokohama City

University, 641-12 Maioka-cho, Totsuka-ku, Yokohama,

Kanagawa244-0813, Japan

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

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