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ISSN : 2287-5824(Print)
ISSN : 2287-5832(Online)
Journal of The Korean Society of Grassland and Forage Science Vol.43 No.1 pp.42-49
DOI : https://doi.org/10.5333/KGFS.2023.43.1.42

Arabidopsis Transcription Factor ANAC032 Enhances Salinity and Drought Tolerance

Netty Ermawati1, Sang Gon Kim2, Joon-Yung Cha3*, Daeyoung Son4*
1Department of Agricultural Production and Central Laboratory for Biosciences, State Polytechnic of Jember, Jember 68101, Indonesia
2Gyeongnam Anti-Aging Research Institute, Sancheong 52215, Republic of Korea
3Dvision of Applied Life Science (BK21 four), Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Republic of Korea
4Department of Plant Medicine, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea

†These authors equally contributed this work.


* Corresponding author: Joon-Yung Cha, Dvision of Applied Life Science (BK21 four), Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Republic of Korea Daeyoung Son, Department of Plant Medicine, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea Tel: +82-55-772-0092, jycha@gnu.ac.kr (JYC) and dyson@gnu.ac.kr (DS)
March 20, 2023 March 27, 2023 March 27, 2023

Abstract


The plant-specific NAC transcription factors control various biological processes, including plant development and stress responses. We have isolated an ANAC032 gene, one of the NAC transcription factor family, which was highly activated by multi-abiotic stresses, including high salt and drought in Arabidopsis. Here, we generated transgenic plants constitutively expressing ANAC032 and its knockout to identify the functional roles of ANAC032 in Arabidopsis under abiotic stress responses. The ANAC032-overexpressing plants showed enhanced tolerance to salinity and drought stresses. The anac032 knockout mutants were observed no significant changes under the high salt and drought conditions. We also monitored the expression of high salt and drought stress-responsive genes in the ANAC032 transgenic plants and anac032 mutant. The ANAC032 overexpression upregulated the expression of stress-responsive genes, RD29A and ERD10, under the stresses. Thus, our data identify that transcription factor ANAC032 plays as an enhancer for salinity and drought tolerance through the upregulation of stress-responsive genes and provides useful genetic traits for generating multi-abiotic stress-tolerant forage crops.


ANAC032 , Drought stress , NAC transcription factor , Salt stress

초록

    Ⅰ. INTRODUCTION

    Osmotic stress is cellular stress induced by extreme environmental conditions, such as drought and high salt levels, which adversely affect to plant growth and development (Chen and Jiang, 2010). Salinity and drought are two major abiotic stresses that limit the production of food crops worldwide (Khalid et al., 2023). They induce various biochemical and physiological changes in plants, which respond and adapt to survive (Hetherington and Waterhouse, 2002;Shinozaki and Dennis, 2003). Several genes have been studied that respond to drought or salt stress at the transcriptional level (Guo et al., 2002;Seki et al., 2002;Xiong et al., 2002). In addition, various genes encoding transcription factors (TFs) were found to be involved in stress responses, suggesting that different transcriptional regulatory mechanisms function in the stress signaling pathways (Shinozaki and Yamaguchi-Shinozaki, 2007).

    NAC (NAM, ATAF1, ATAF2, CUC2)-domain proteins are one of the largest families of plant-specific TFs, and are known to control multiple processes, including plant developments (Souer et al., 1996;Duval et al., 2002), defense (Bu et al., 2008;Jensen et al., 2008), hormones (Xie et al., 2000; He et al., 2002), membrane-associated cell cycle (Kim et al., 2006), and stress responses (Hegedus et al., 2003;Fujita et al., 2004;Tran et al., 2004;Lu et al., 2007). Other members of ATAF subgroup, ArabidopsisNAC019 (ANAC019), ANAC055, and ANAC072 genes have been shown significantly increased drought tolerance in overexpression plants (Tran et al., 2004;Bu et al., 2008). Fujita et al. (2004) showed that drought and high salinity stresses enhance ANAC072 transcripts. Furthermore, ATAF1 and LOV1 are drought-inducible genes that negatively regulate the expression of stress-responsive genes and control flowering time and cold response in Arabidopsis, respectively (Lu et al., 2007;Yoo et al., 2007). Recently, we have screened two multi-abiotic stress-responsive NAC TFs, ANAC032, and ANAC083, through microarray analysis and identified their transcriptional levels, subcellular localization, and promoter activity under various stresses (Ermawati et al., 2021).

    In this study, we generated ANAC032-overexpressing transgenic Arabidopsis plants and analyzed stress responses compared with ANAC032 knockout mutant (anac032) plants. We found that ANAC032-overexpressing plants showed enhanced tolerance to high salt and drought stress with higher root growth and survival rate, respectively. Thus, our findings support that ANAC032 positively involves multi-abiotic stress responses, such as drought and salt stresses.

    Ⅱ. MATERIALS AND METHODS

    1. Plant materials and stress treatments

    Arabidopsis thaliana (ecotype Columbia) were grown on MS medium agar plates under a 16 h light (100 μmol photons m-2 s-1) /8 h dark condition. For phenotypic analysis under different stress conditions, Arabidopsis seedlings were transferred to the MS liquid medium with or without 150 mM NaCl or 300 mM mannitol. For drought stress, the plants grown in soil were withheld watering for 10 days. For Northern blot analysis, plants were applied with 300 mM NaCl (to monitor the rapid induction of stress-responsible genes) or 300 mM mannitol for indicated time points.

    2. Generation of ANAC032-overexpressing plants and anac032 knockout mutants

    The ANAC032 cDNA was amplified from Arabidopsis RNA by RT-PCR using full-length primers for ANAC032 (Table 1). The PCR products were digested with BamH I/Pst I for ANAC032 and then cloned into the pCAMBIA 1300PT vector under the control of 35S promoter for overexpression. Agrobacterium strain GV3101 containing this binary construct was transformed into the Arabidopsis using the floral dipping method (Clough et al., 1998). Transformants were selected on MS medium containing hygromycin (30 mg/L). An ArabidopsisANAC032 T-DNA insertion lines (SALK_087702 and SALK_012253) were obtained from the Arabidopsis Biological Resource Center (Columbus, OH). The T-DNA insertion sites were confirmed by PCR using T-DNA left border primer and ANAC032 reverse primer for anac032 knockout mutants (Table 1).

    3. Determination of total chlorophyll

    To measure the total chlorophyll, 100 mg of 3-week-old leaf tissues were collected and extracted with 8 mL of 80% (v/v) acetone for 24 h. The absorptions of the extracts were determined at 645 and 663 nm using the UV/Visible Spectrophotometer (Pharmacia LKB). The concentrations of total chlorophyll were calculated according to a method previously described (Arnon, 1949).

    4. Gene expression analysis

    Total RNA from untreated and stress-treated Arabidopsis seedlings was extracted using the phenol/LiCl method (Ermawati et al., 2021). Total RNA (20 μg) was fractionated on 1.2% (w/v) formaldehyde agarose gel and blotted on Hybond-N nylon membrane (Amersham) with 10x SSC. Blots were hybridized overnight at 65°C with a 32P-labeled full-length DNA using the Megaprime DNA labeling system (Amersham). After hybridization, the blots were washed twice with 2x SSC containing 1% (w/v) SDS for 15 min at room temperature and twice with 0.1x SSC containing 0.5% SDS for 10 min at 65°C. The blots were exposed to X-ray film (Fuji Photo Film).

    Ⅲ. RESULTS AND DISCUSSION

    1. Generation and phenotypic analysis of ANAC032-overexpressing plants and anac032 knockout mutants

    To verify the functional roles of ANAC032 in plants, we analyzed stress responses and found that transcription of ANAC032 was induced under multi-abiotic stress conditions (Ermawati et al., 2021). Thus, to confirm the enhanced gene expression levels of ANAC032 under diverse abiotic stress conditions in planta, we generated transgenic plants having constitutive expression of ANAC032 driven by 35S promoter (Fig. 1A). In addition, we screened two independent loss-of-function mutant plants of ANAC032 (anac032-1 and anac032-2) with T-DNA insertion (Fig. 1B), and the expression levels of ANAC032 were confirmed in two independent lines of both ANAC032-overexpressing and anac032 plants (Fig. 1C). In phenotypic analysis, the ANAC032-overexpressing transgenic Arabidopsis plants had a yellowish leaves compared to those of wild-type (WT) and anac032 (Fig. 1D). In addition, the phenotypes of independent lines of anac032 showed no differences from the WT (Fig. 1D). We further analyzed the total chlorophyll contents in WT, ANAC032-overexpressing, and anac032 plants (Fig. 1E). The total chlorophyll contents in ANAC032-overexpressing plants were approximately 35% lower than those in WT, while the contents in anac032 mutants were almost similar to the WT (Fig. 1E). The yellowish leaves in ANAC032-overexpressing plants were not relate to leaf senescence, because the yellowish leaves gradually decreased and turned greenish in 4-week-old plants. The yellowing leaf like as leaf senescence is likely to be induced by various factors including nutrients, light, and sugar (van Doorn, 2008), and thus ANAC032 may temporally inhibit chlorophyll biosynthesis in the juvenile phage.

    2. ANAC032-overexpression enhanced salinity and drought tolerance.

    To verify whether overexpression of ANAC032 induces tolerance to abiotic stresses, salt (150 mM NaCl) and osmotic stress (300 mM mannitol) were applied to WT, ANAC032- overexpressing plants, and anac032 mutants (Fig. 2). After exposure in high concentration of NaCl for 8 days, ANAC032-overexpressing plants were enhanced tolerance to the salt stress with high biomass of aerial and root parts compared to WT (Fig. 2A). The ANAC032-overexpressing plants also showed tolerance to mannitol-induced osmotic stress (Fig. 2A). However, no phenotypic changes in anac032 mutants were observed under high salt and osmotic stress compared to the WT (Fig. 2B).

    ANAC032-overexpressing plants produced more lateral roots than the WT under control and high salt stress conditions. This phenotype was similar to that observed in Arabidopsis NAC1 (Xie et al., 2000) and Brassica napus NAC (Hegedus et al., 2003), which were also greatly affected by high salinity and drought. Thus, we examined whether the ANAC032-overexpressing plants have enhanced tolerance to drought stress. In order to make the stress conditions homogenous, we first measured the soil water content by weighing the soil in the pots including the plants which grew on those pots. When the water contents were held in soil under 50%, the drought stress was conducted by withholding from watering. During the drought stress treatment for 10 days, all plants withered completely (Fig. 3). After re-watered for 4 days, the survival rate of ANAC032-overexpressing plants were 87%, which was higher than WT (60%) and anac032 mutant (51%) (Fig. 3). Thus, these results indicate that overexpression of ANAC032 enhances salinity and drought tolerance in Arabidopsis.

    3. Overexpression of ANAC032 activates the upregulation of high salt and drought stressresponsive RD29A and ERD10 genes.

    Next, we investigated whether changes in the ANAC032 expression triggers alteration of stress-responsive genes expression through high salt and drought stress signaling pathway. Northern blot analysis showed the expression of ANAC032 was significantly increased by high salt and mannitol treatments in a time-dependent manner in WT (Fig. 4). The ANAC032 was constitutively expressed irrespectively of stress treatments in the ANAC032-overexpressing plants (Fig. 4). We further monitored the changes in expression of stress-responsive RD29A and ERD10 genes, which have been reported to be induced by high salt and drought stresses (Yamaguchi-Shinozaki and Shinozaki, 1993;Kasuga et al., 1999;Zhu, 2002). Under high salt conditions, the transcript levels of RD29A and ERD10 in the WT increased at 1 h to peak levels at 6 h, and decreased thereafter (Fig. 4A). In the ANAC032-overexpressing plants, the expressions of both stress genes were higher than in WT, however, the expression in anac032 mutants was decreased under salt stress conditions compared to WT and ANAC032-overexpressing plants (Fig. 4A). Upon mannitol treatment, ANAC032 overexpression caused significant upregulation of RD29A and ERD10 compared to WT and anac032 mutants (Fig. 4B). However, the expression levels of ERD10 under mannitol treatments showed no significant difference between the WT and anac032 mutants (Fig. 4B). Thus, these results suggest that the upregulation of ANAC032 activates RD29A and ERD10 inductions by salt and drought stress, thereby enhancing tolerance to the multi-abiotic stresses in Arabidopsis.

    Ⅳ. CONCLUSION

    The NAC family is one the largest TF families in plant genomes and involves in diverse plant developments and stress responses. Recently, we have identified using microarray analysis that the ArabidopsisANAC032 is one of the NAC TFs highly induced by various abiotic stresses (Ermawati et al., 2021). In this study, functional analysis was performed by overexpression or knock-out of ANAC032 in Arabidopsis, and data showed that overexpression of ANAC032 enhanced tolerance to salinity and drought through upregulation of RD29A and ERD10. Taken toget her, these findings reveal a novel function of ANAC032, a NAC TF, as an enhancer for salinity and drought tolerance, and provide informative adaptive strategies for overcoming food crops production limitations caused by salinity and drought.

    Ⅴ. ACKNOWLEDGEMENTS

    This study was conducted with support from the Rural Development Administration (PJ014155042022).

    Figure

    KGFS-43-1-42_F1.gif

    Generation of ANAC032-overexpressing and knockout mutant plants and phenotypic analysis. Schematic diagram of ANAC032-overexpression construct (A) and T-DNA insertion sites in its knockout mutant (anac032) plants (B). (C) Confirmation of ANAC032 gene expression in ANAC032-overexpressing and anac032 plants. The expression of tubulin was used as an internal control. (D) Phenotypic analysis. Three-week-old of Arabidopsis wild-type, ANAC032 -overexpressing, and anac032 plants grown in soil were pictured. (E) Chlorophyll contents. Three-week-old wild-type, ANAC032-overexpressing, and anac032 plants were used for chlorophyll contents. Bars show means±SD values of four independent experiments. Significant differences are shown as asterisks (Student t-test, *p<0.05; ***p<0.001; n.s., no significant).

    KGFS-43-1-42_F2.gif

    The overexpression of ANAC032 enhances tolerance to high salt and hyperosmotic stress. Seven-day-old seedlings of Arabidopsis wild-type (WT), ANAC032-overexpressing, and anac032 plants grown on MS medium plates were transferred to medium with or without 150 mM NaCl (A) and 300 mM mannitol (B) and grown for another 8 and 5 days, respectively.

    KGFS-43-1-42_F3.gif

    The overexpression of ANAC032 enhances tolerance to drought stress. (A) Drought stress phenotypes. Drought stress treatment was conducted in 5-week-old Arabidopsis wild-type (Col WT), ANAC032-overexpressing, and anac032 plants by withholding water for 10 days, and then re-watered for another 4 days and pictured. (B) Survival rate. The number of survived plants was counted and normalized compared to the total plants examined. Data represent the mean±SD (n=3); n refers to the number of biological replicates. Significant differences are shown as asterisks (Student t-test, *p<0.05; ***p<0.001; n.s., no significant).

    KGFS-43-1-42_F4.gif

    The overexpression of ANAC032 activates stress-responsive gene expressions in response to high salt and hyperosmotic stress. Arabidopsis wild-type (WT), ANAC032-overexpressing (ANAC032#8), and ANAC032 knockout mutant (anac032-1) plants treated with 300 mM NaCl (A) or 300 mM mannitol (B) for the indicated times were subjected to Northern blot analysis.

    Table

    List of oligonucleotide primers

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