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ISSN : 2287-5824(Print)
ISSN : 2287-5832(Online)
Journal of The Korean Society of Grassland and Forage Science Vol.41 No.4 pp.242-249
DOI : https://doi.org/10.5333/KGFS.2021.41.4.242

Isolation of Multi-Abiotic Stress Response Genes to Generate Global Warming Defense Forage Crops

Netty Ermawati1, Jong Chan Hong2, Daeyoung Son3*, Joon-Yung Cha4*
1Department of Agricultural Production and Central Laboratory for Biosciences, State Polytechnic of Jember, Jember 68101, Indonesia
2Division of Life Science, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju
52828, Republic of Korea
3Department of Plant Medicine, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea
4Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Republic of Korea
* Corresponding author: Daeyoung & Joon-Yung, Tel: +82-55-772-1926 (DS); +82-10-4848-6144 (JYC), Fax: +82-55-772-1929, E-mail: dyson@gnu.ac.kr (DS) & jycha@gnu.ac.kr (JYC)
November 7, 2021 November 23, 2021 November 23, 2021

Abstract


Forage crop management is severely challenged by global warming-induced climate changes representing diverse a/biotic stresses. Thus, screening of valuable genetic resources would be applied to develop stress-tolerant forage crops. We isolated two NAC (NAM, ATAF1, ATAF2, CUC2) transcription factors (ANAC032 and ANAC083) transcriptionally activated by multi-abiotic stresses (salt, drought, and cold stresses) from Arabidopsis by microarray analysis. The NAC family is one of the most prominent transcription factor families in plants and functions in various biological processes. The enhanced expressions of two ANACs by multi-abiotic stresses were validated by quantitative RT-PCR analysis. We also confirmed that both ANACs were localized in the nucleus, suggesting that ANAC032 and ANAC083 act as transcription factors to regulate the expression of downstream target genes. Promoter activities of ANAC032 and ANAC083 through histochemical GUS staining again suggested that various abiotic stresses strongly drive both ANACs expressions. Our data suggest that ANAC032 and ANAC083 would be valuable genetic candidates for breeding multi-abiotic stress-tolerant forage crops via the genetic modification of a single gene.


ANAC032 , ANAC083 , Multi-abiotic stress , NAC transcription factor

초록

    Ⅰ. Introduction

    Forage crop production and grassland are essential for managing livestock industries; however, the production is widely challenged by global warming (Wheeler and Reynolds, 2013). As forage crop cultivation is based on outdoor farming, crops are primarily affected by environmental factors such as drought, high salinity, and extreme temperature such as hot and cold temperatures. Plants respond and adapt through various biochemical and physiological processes to overcome these environmental stresses by the transcriptional activation of diverse gene sets encoding heat-shock proteins, antioxidants, ion transporters, and so on (Bray et al., 2000). Extensive screening of genes responding to various abiotic stresses has been applied in the model plant Arabidopsis and in various forage crops (Lee et al., 2018;Tawab et al., 2020). Thus, the genetic modification will be helpful to develop forage crops defending to current dynamic climate changes.

    The stress signaling pathways mediating these adaptations are controlled mainly by transcription factors (TFs) and cis-regulatory elements (Shinozaki et al., 2000). TFs are proteins that respond to environmental stimuli through a signaling cascade and bind to specific regulatory sites upstream of constituent genes in a regulatory network by direct physical interaction or in combination with other proteins. Consequently, an alteration of the expression of transcription factor genes results in dramatic differences in the expression of multiple genes in plants (Liu et al., 1999). The cis-regulatory elements of some genes that have a typical stress-inducible expression profile and the transcription factors that affect the expression of these genes have been identified (Shinozaki and Yamaguchi-Shinozaki, 2007). One of the most prominent families of plant-specific transcription factors is NAC proteins (Riechmann et al., 2000). NAC (NAM, ATAF1, ATAF2, CUC2)-domain proteins are a class of TFs known to control multiple processes, including plant development, defense, and hormone and stress responses (Riechmann et al., 2000). NAC genes have also been involved in light responses, programmed cell death, senescence, and various abiotic stresses (Shao et al., 2015). Although efficient transformation techniques have been developed in forage crops (Lee et al., 2014), identifying valuable genetic materials responding to multi-abiotic stresses will be a fast-approaching strategy to develop the forage crops.

    In this study, we 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.

    Ⅱ. Materials and Methods

    1. Plant materials and stress treatments

    Arabidopsis thaliana (ecotype Columbia) were grown on germination medium (GM) agar plates under a 16 h light/8 h dark condition. For microarray analysis, mRNA treated with salt, drought, and cold stresses for 3 h were labeled with Cy5 and Cy3 (for untreated) fluorescent probes. The cDNA probes were hybridized with the cDNA microarray prepared from ~1300 full-length Arabidopsis cDNAs on the DNA chip. For treatment under different stress conditions, 7-day-old seedlings were transferred to the MS liquid medium with or without 100 μM abscisic acid (ABA), 250 mM (for Northern blot analysis) or 200 mM NaCl (for semi-quantitative RT-PCR and histochemical analyses), 20% (v/v) polyethylene glycol (PEG) 6000 or 200 mM mannitol then incubated in the culture room by shaking for indicated time. For dehydration and cold treatments, plants were incubated on 3 MM paper for 30 min and placed in a 4 ℃ chamber for 24 h, respectively. For wounding stress treatment, half of the rosette leaves were scratched with a sterile needle and maintained for 1 day.

    2. Northern blot analysis

    Total RNA from untreated and stress-treated Arabidopsis seedlings was extracted using the phenol/LiCl method (Ausubel et al., 2003). Total RNA (20 μg) was fractionated on 1.2% formaldehyde agarose gel and blotted on Hybond-N nylon membrane (Amersham) with 10x SSC. Blots were hybridized overnight at 65 ℃ 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 ℃. The blots were exposed to X-ray film (Fuji Photo Film).

    3. Semi-quantitative RT-PCR analysis

    RNA was extracted from wild-type Arabidopsis plants using the Trizol reagent (Invitrogen). Five micrograms of RNA of each genotype with 1 μg oligo (dT) primer were heated at 70 ℃ for 5 min and then chilled on ice immediately. RNA was then subjected to RT with RevertAidTM M-MuLV reverse- transcriptase (Fermentas) at 42 ℃ for 1 h according to the manufacturer’s protocol. Synthesized cDNA was used as a template for PCR. The PCR amplify of expected genes was conducted with the following primers in Table 1, and the tubulin gene was used as a loading control.

    4. ANAC032- and ANAC083-GUS promoter constructs

    The promoter regions (1500 bp upstream from the start codon) of the ANAC032 and ANAC083 genes were PCR-amplified with the following primer pair for GUS-expression in Table 1. The PCR fragment was inserted into entry vector pDONR221 and then recombined into the destination vector pBGWFS7 by Gateway methods (Invitrogen). These ANAC032 and ANAC083 promoter-GUS constructs were introduced into Agrobacterium tumefaciens strain GV3101 and transformed into wild-type Arabidopsis. T1 transgenic lines resistant to Basta herbicide (glufosinate) were used for generations. T2 transgenic lines were selected on MS medium containing phosphinothricin (4 mg ml-1).

    5. Histochemical GUS assay

    For GUS assay, T3 seedlings grown on MS agar plates were was incubated in a solution containing 50 mM phosphate buffer, 10 mM EDTA (pH 8), 0.1% (v/v) Triton X-100, 1.25 mM potassium ferricyanide, 1.25 mM potassium ferrocyanide, and 0.1% (w/v) 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-Gluc) for 12 h at 37 ℃ and then washed with ethanol to remove chlorophylls.

    6. In vivo targeting of GFP fusion constructs

    Subcellular localization analysis of ANAC032 and ANAC083 was prepared using leaf tissues of 10-day-old Arabidopsis plants grown on MS in the growth chamber. Leaves were cut with a razor blade and incubated with 30 ml of enzyme solution containing 1% (w/v) macerozyme R-10 (Yakult Honsha Co), 4% (w/v) cellulase onozuka R-10 (Yakult Honsha Co), 0.1% (w/v) pectolyase Y23 (Yakult Honsha Co), 400 mM mannitol, 1 mM CaCl2, and 0.5% (w/v) BSA (pH 5.6) at 22 ℃ for 24 h with gentle agitation (50 to 75 rpm). To construct ANAC032::GFP and ANAC083::GFP, cDNA was amplified with the primer pairs F032GFP and R032GFP for ANAC032, and primer pairs F083GFP and R083GFP for ANAC083 (Table 1) by deletion of the stop codon. The PCR fragments were then fused into pUC19 modified vector (smGFP) driven by 35S promoter. The construct was transformed to protoplast using polyethylene glycol-mediated transformation. The images were monitored and captured by IX71 microscope camera (Olympus). The filters sets used are U-MGFPHQ mirror units (exciter, 460~480HQ: dichroic, 485: and emitter, 495~540HQ).

    Ⅲ. RESULTS AND DISCUSSION

    1. Isolation of multi-abiotic stresses responsible genes by microarray analysis.

    Microarray analysis was applied to isolate multiple abiotic stress responsive genes against significant environmental stresses such as salt, drought, and cold. Over 500 genes among 1300 TFs prepared on DNA chip were commonly induced by these treatments. We regarded genes with an expression ratio greater than three-folds (3 h salt vs. untreated, 3 h drought vs. untreated, or 3 h cold vs. untreated) as multi-stress-inducible genes (Fig. 1A) (Ooka et al., 2003). For further functional analysis in abiotic stress responses, two TFs (ANAC032 and ANAC083) genes were selected. NAC TFs including ORESARA1 (ORE1)/ANAC092 are well characterized in leaf senescence as positive regulators (Kim et al., 2009;Yang et al., 2011;Mahmood et al., 2016). However, our two NAC TFs are not well characterized yet under diverse abiotic stresses in detail.

    Expression patterns of ANAC032 and ANAC083 were validated using Northern blot analysis. NaCl (250 mM) treatment as salt stress, PEG 6000 (20%) treatment as alternative drought stress, and ABA treatment (100 μM) as a stress-inducible phytohormone, and cold stress were applied to Arabidopsis seedlings and examined transcriptional levels with their representative marker genes such as RD29A for salt, drought and ABA treatments and Cor15A for cold treatment. Both ANAC genes were gradually increased by NaCl, ABA, or cold treatment and peaked at 1 h of PEG 6000 treatment (Fig. 1B). Thus, ANAC032 and ANAC083 could be positive regulators and candidates for developing multi-abiotic stress-responsive forage crop plants.

    2. The transcriptional pattern of ANAC032 and ANAC083.

    To confirm whether expression of ANAC032 and ANAC083 genes is affected by abiotic stresses, seven-day-old seedlings of Arabidopsis wild-type were subjected to various abiotic stresses. Quantitative PCR analyses showed that transcript levels of ANAC032 and ANAC083 genes in wild-type plants were highly increased by ABA, high salt, or drought treatments (Fig. 2A, C). The increasing expressions were 2-fold over than non-treated plants. Cold, osmotic stress or wounding treatments also highly induced the transcript levels of ANAC032 and ANAC083 (Fig. 2A, C).

    To obtain further clues about their functions, we analyzed the expression pattern of ANAC032 and ANAC083 in different tissues. As shown in Fig. 2B, the ANAC032 gene was expressed in silique, leaves, and roots, mature plant tissues. Moreover, ANAC083 was highly induced in both cauline and rosette leaves (Fig. 2D). These accelerated expression patterns in leaves may be correlated with leaf longevity as positive regulators for delaying leaf senescence (Yang et al., 2011;Mahmood et al., 2016).

    3. Subcellular localization of ANAC032 and ANAC083.

    TFs control transcriptional regulation in massive biological processes in plant development and environmental stresses (Ooka et al., 2003;Chapman and Estelle, 2009). TFs are localized in the nucleus and regulate their expression of downstream target genes via their DNA-binding motifs. ANAC032 protein contains the NAC-domain, divided into 5 subdomains and has high similarity with other members of the NAC family. However, the C-terminal region shows no significant similarity with other members of NAC family. A putative nuclear localization signal (NLS) sequence (PRDRKYPNGS) was found in its N-terminal region. The closest relative of ANAC032 is ATAF1, with which it shares around 65% amino acid similarity (data not shown). ATAF1 is involved in dehydration stress and ABA-dependent (Lu et al., 2007). In addition, the N-terminal region of ANAC083 shares a high identity to other NAC domain-containing proteins and a variable C-terminal domain. The five motifs (A to E) comprising the NAC domain presented in the N-terminal region. A putative NLS sequence (AKYPNGN) of ANAC083 was predicted in the third motif of NAC domain.

    Thus, we constructed GFP-fused ANAC032 and ANAC083 constructs driven by 35S promoter and confirmed their subcellular localization in Arabidopsis protoplasts. Both ANAC032 and ANAC083 were presumably targeted to nuclei (Fig. 3). The merged images of ANACs::GFP with a positive marker, NLS-RFP (a control for nuclear localization), clearly showed nuclear localization (Fig. 3). These implied that the general characteristics of NAC proteins are nuclear proteins, and function as TFs for regulating transcription of downstream genes.

    4. Promoter activity of ANAC032 and ANAC083.

    To identify more detail, the temporal and spatial expression patterns of ANAC032 and ANAC083 were determined by histochemical β- glucuronidase (GUS) staining of transgenic plants that harbored an ANAC032 or ANAC083 promoter-GUS construct. With the ANAC032 or ANAC083 promoter (1.5 Kb from start codon) construct, GUS activity was monitored at various growth stages of seedlings. First, GUS activity of ANAC032 was constitutively expressed in 2-, 5- and 7-day-old seedlings (data not shown). However, its activity was not detected in 2-week-old seedlings. Interestingly, treatment with exogenous ABA, NaCl or mannitol in 2-week-old seedlings for 12 h enhanced the ANAC032 promoter activity, mainly in the roots (Fig. 4A). Wounding treatment was not induced the expression of ANAC032 promoter (data not shown), even though in the promoter region of ANAC032 consists of six recognition sites for WUN boxes and two sites for W-boxes (Fig. 4B), which are known as recognition sites in response to wounding and pathogen attack, respectively (Pastuglia et al., 1997;Maleek et al., 2000). Other cis-elements found in the promoter sequence of ANAC032 using Plant CARE software http://bioinformatics. psb.ugent.be/webtools/plantcare/html/) are six ABA-responsive elements (ABRE) and one dehydration responsive element (DRE), known as elements response in drought and salt stresses (Shinozaki et al., 2003). An as-1 motif is also present in the promoter, known to be oxidative stress-responsive and hypersensitive responsive elements (Lam et al., 1989;Selth et al., 2005). Second, the spatial and temporal expression pattern of ANAC083 was also determined by GUS staining of transgenic plants harboring an ANAC083 promoter-GUS reporter construct. In germination stages, GUS activity of ANAC083 promoter was constitutively expressed in 2-, 5- and 7-day-old seedlings (data not shown). In 2-week-old seedlings, the GUS expression in leaves and roots was very low. However, the GUS expression in seedlings treated by ABA, NaCl, or mannitol increased mainly in the roots (Fig. 4C). These results indicated that the upstream sequence present in ANAC083 contains sufficient cis-regulatory elements to respond to ABA, NaCl, or osmotic treatment. In silico analysis showed that the upstream sequence (1.5 Kb) of ANAC083 contains ABRE, WUN, and As1- elements, which are involved in ABA, wounding, and oxidative responses (Fig. 4D) (Yamaguchi-Shinozaki and Shinozaki, 1994).

    Ⅳ. CONCLUSION

    Although massive genetic resources responding to various environmental stresses have been identified, most of them are restricted in a single abiotic stress response. However, current global warming-induced climate changes are complex due to the regions and nations. Thus, multi-abiotic stresses responsive genetic materials are urgently required for developing stresstolerant crops. TFs regulate the transcriptional expression of diverse downstream target genes via direct association with the chromosome. This study found two ANAC genes induced by multi-abiotic stresses including salt, drought and cold stresses by microarray analysis. Both ANAC genes were localized in the nucleus. Histochemical analysis revealed that various abiotic stresses activated their promoters of ANAC032 and ANAC083. Taken together, the heterologous expression of ANAC032 and ANAC083 in forage crops would enhance stress tolerance against multi-abiotic stresses.

    Ⅴ. ACKNOWLEDGEMENTS

    This research was funded by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT 2019R1A2C1011173).

    Figure

    KGFS-41-4-242_F1.gif
    Microarray preparation and Northern blot analysis. (A) A schematic performing of microarray analysis for multi abiotic stress inducible genes. (B) Conformation of isolated ANAC genes under abiotic stresses by Northern blot analysis. Seven-day-old Arabidopsis wild-type seedlings were exposed to NaCl (250 mM), PEG (20%), ABA (100 μM), or cold (4˚C) for indicated time period.
    KGFS-41-4-242_F2.gif
    The expression of ANAC genes in wild-type plants. Semi-quantitative RT-PCR analyses of ANAC032 (A and B) and ANAC083 (C and D) transcripts. Seven-day-old Arabidopsis Col-0 seedlings were treated as follows, control (no treatment), ABA (100 μM) for 12 h, NaCl (200 mM) for 12 h, drought as dehydration on 3MM paper for 1 h, cold (4 °C) for 24 h, mannitol (200 mM) for 12 h, and wounding. Data represent means±SD of three independent experiments.
    KGFS-41-4-242_F3.gif
    In vitro targeting of the GFP-fused ANAC032 or ANAC083 proteins. The GFP::ANAC032 (A) or GFP::ANAC083 (B) fusion protein was introduced into Arabidopsis protoplast. NLS-RFP was used as a nuclear marker.
    KGFS-41-4-242_F4.gif
    Histochemical analysis of ANAC032 and ANAC083 promoter-GUS. GUS staining of transgenic plants harboring ANAC032 (A) or ANAC083 (B) promoter-GUS. Two-week-old plants were subjected to distilled water as a control, ABA (100 μM), NaCl (200 mM) or mannitol (200 mM) for 12 h. Distribution of cis-acting element in the promoter region of ANAC032 (B) and ANAC083 (D).

    Table

    List of oligonucleotide primers used in this study

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