Novel Maize NAC Transcriptional Repressor ZmNAC071 Confers

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Novel Maize NAC Transcriptional Repressor ZmNAC071 Confers Enhanced Sensitivity to ABA and Osmotic Stress by Downregulating Stress-Responsive Genes in Transgenic Arabidopsis Lin He,† Jing Bian,† Jingyu Xu,* and Kejun Yang* Key Laboratory of Modern Agricultural Cultivation and Crop Germplasm Improvement of Heilongjiang Province, Heilongjiang Bayi Agricultural University, 5 Xinfeng Road, 163319 Daqing, China Downloaded via UNIV PARIS-SUD on August 5, 2019 at 19:42:17 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: NAC TFs play crucial roles in response to abiotic stresses in plants. Here, ZmNAC071 was identified as a nuclear located transcriptional repressor. Overexpression of ZmNAC071 in Arabidopsis enhanced sensitivity of transgenic plants to ABA and osmotic stress. The expression levels of SODs, PODs, P5CSs, and AtMYB61 were inhibited by ZmNAC071, which results in reduced ROS scavenging and proline content, increased ROS level, and water loss. Besides, the expression levels of some ABA or abiotic stress-related genes, like ABIs, RD29A, DREBs, and LEAs were also significantly inhibited by ZmNAC071. Yeast one-hybrid assay demonstrated that ZmNAC071 specifically bound to the cis-acting elements containing CGT[G/A] core sequences in the promoter of stress-related genes, suggesting that ZmNAC071 may participate in the regulation of transcription of these genes through recognizing the core sequences CGT[G/A]. These results will facilitate further studies concerning the cis-elements and downstream genes targeted by ZmNAC071 in maize. KEYWORDS: NAC transcriptional repressor, maize, osmotic stress, ABA, reactive oxygen species, proline biosynthesis

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INTRODUCTION Abiotic stresses such as salt, drought, and low temperature can impose osmotic stress on crops.1 To survive, plants have developed multifaceted strategies at different levels to adapt to environmental cues.2 At the molecular levels, transcription factors (TFs) are considered to be pivotal components of the abiotic stress signal transduction pathway because they can control the expression of a series of stress-related genes via interacting with cis-elements.3 NAC (NAM, ATAF1/2, and CUC2) is one of the largest plant-specific TF families. Members of this gene family contain a highly conserved N-terminal DNA-binding domain and a diversified C-terminal domain.4 NAC TFs are involved not only in the regulation of growth, development, metabolism, and biotic stress response but also in the regulation of abiotic stress response in plants.5−9 NAC TFs function as transcriptional activators or repressors via upregulating or downregulating the expression of downstream target genes during abiotic stresses, leading to stress-tolerant or stress-sensitive phenotypes. In Arabidopsis, ANAC019, ANAC055, and RD26 (RESPONSIVE TO DEHYDRATION 26) positively regulate abiotic responses,10,11 while NAC016, AtNAP, and ANAC069 negatively regulate abiotic responses.12−14 In rice, overexpression of ONACO22 or OsNAC045 enhanced resistance of transgenic plants to both drought and salt.15,16 Similarly, overexpressing root-specific NAC TFs OsNAC9 and OsNAC10 significantly increased yield of transgenic rice under field drought conditions.17,18 However, overexpression of miR164targeted NAC genes (OMTN2, OMTN3, OMTN4, and OMTN6) reduced drought tolerance of transgenic rice at the reproductive stage.19 In maize, only a few NAC members implicated in the regulation of stress signal transduction © XXXX American Chemical Society

pathway were identified. ZmSNAC1, ZmNAC55, and ZmNAC84 conferred drought tolerance in transgenic Arabidopsis or tobacco.20−22 Overexpression of ZmNAC111 improved drought resistance at the seedling stage in both Arabidopsis and maize.23 However, to date, there has been no report of maize NACs functioning as transcriptional repressors to negatively regulate abiotic stress responses. Under abiotic stresses, the ROS levels in plant cells can be enhanced by stress-stimulated physiological imbalance.24 To adjust ROS redox homeostasis in cells under adverse environmental conditions, plants have evolved complex regulatory pathways.25 NAC TFs can play an important role in the regulation of ROS metabolism under abiotic stresses. For example, SNAC3 acts as a positive regulator of heat tolerance via regulating the expression of ROS-related genes in rice.26 CsATAF1 improves drought resistance in cucumber through increasing the activity of antioxidant enzymes such as SOD, POD, and CAT.27 BpNAC012 functions as a positive regulator in response to salt and osmotic stresses in Betula platyphylla via upregulating the expression of SODs and PODs.28 Arabidopsis ANAC069 negatively regulates abiotic stress resistance by inhibiting the expression of ROS-associated genes.14 However, it is unclear whether NACs from maize are also implicated in ROS metabolism under abiotic stresses. In the present work, a novel transcriptional repressor gene ZmNAC071 was isolated from maize inbred line He344. Overexpression of ZmNAC071 enhanced the sensitivity of Received: April 13, 2019 Revised: July 23, 2019 Accepted: July 29, 2019

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DOI: 10.1021/acs.jafc.9b02331 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

days to assess root lengths and fresh weights. To study the sensitivity of plants to ABA and osmotic stress at the adult stage, 4-week-old soilgrown plants of WT, OE1, and OE3 were treated with deionized water (control), 50 μM ABA, and 300 mM mannitol for a total 16 days, and then photos were taken. Survival rates were determined after mannitol treatments, and chlorophyll contents were measured after ABA treatments. The results were assessed based on three biological replicates. Physiological Roles of ZmNAC071. Four-week-old plants were treated with deionized water (control), 300 mM mannitol, and 50 μM ABA for 48 h. The rosette leaves were collected for physiological parameters measurement. The contents of H2O2 and O2− were measured using a previously described method by Velikova et al. (2000) and Able et al. (1998), respectively.32,33 The activities of SOD and POD were determined as described by Wang et al. (2012).34 The contents of proline and MDA were measured according to Bates et al. (1973) and Zhang et al. (2009), respectively.35,36 For electrolyte leakage analysis, plants were treated as above for 5 days, and then electrolyte leakage was measured using a previously described method by Wang et al. (2017).37 Water loss rates and stomatal apertures were measured according to Mao et al. (2016) and Guo et al. (2017), respectively.21,38 The results were assessed based on three biological replicates. Histochemical Analysis. Four-week-old WT, OE1, and OE3 plants were treated with deionized water (control), ABA (50 μM), or mannitol (300 mM) for 2 h, and then the whole plants were used for staining analysis. DAB, NBT, and H2DCF-DA staining was performed according to Zhang et al. (2011).39 Evans blue staining was performed using a previously described method by Kim et al. (2003).40 For PI staining analysis, 1-week-old plants were transplanted to 1/2 MS medium supplemented with mannitol (300 mM) and ABA (5 μM) for 24 h. Then plants were infiltrated with PI (25 μg/mL) for 30 min. The staining results were monitored by laser scanning confocal microscopy (Leica SP8). Yeast One-Hybrid (Y1H) Assay. The effector vector was obtained by inserting the CDS of ZmNAC071 into the pGADT7Rec2 vector. One tandem copy of the NAC recognition sequence (R1−5) with the core sequence “CACG” (or its reverse complementary sequences “CGTG”) or “CGTA” (or its reverse complementary sequences “TACG”) and mutant core sequence (R6) were fused to pHIS2 vector as the reporter vectors, respectively. The promoters containing at least 2 core sequences were also fused to pHIS2 vector as reporter vectors. The schematic diagram of the truncated promoters is shown in Figure S1. The primers used are listed in Table S1. Y1H assay was performed according to the manufacturer’s protocols (Clontech, USA). The effector vector, together with each reporter vector, was cotransformed into Y187 yeast strain for DNA−protein interaction analysis. The growth performances of cotransformants were examined on DDO (SD/-Leu/-Trp) or TDO (SD/-Leu/-Trp/-His) medium supplied with 3-AT. Statistical Analyses. Statistical analysis was performed using the SPSS 16 software. All values are the mean (±SD) of three biological replicates. Asterisks represent statistical significance compared to control using Student’s t test (*, P < 0.05, **, P < 0.01).

plants to ABA and osmotic stress by inhibiting the expression of SODs, PODs, P5CSs, AtMYB61, ABIs, RD29A, DREBs, and LEAs. The core sequences CGT[G/A] were important for the regulation of ZmNAC071-mediated gene expression. This work improves our understanding of the roles of maize NAC TFs and sensitivity mechanisms underlying plants responding to ABA and osmotic stress.

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MATERIAL AND METHODS

Plant Materials and Stress Treatments. The pregermination treatments of maize (He344 inbred line) seeds were performed as described by Li et al. (2012).29 Then seeds that grew uniformly were transferred into half-strength Hoagland solution to cultivate.30 The growth conditions were set according to Li et al. (2012).29 To analyze the gene expression profiles of ZmNAC071 under abiotic stress, the roots of three-leaf stage seedlings were submerged into half-strength Hoagland’s liquid medium supplemented with 300 mM mannitol for 0, 1, 3, 6, and 12 h or 50 μM ABA for 0, 0.5, 1, 2, and 4 h. The nutrient solution was aerated with an air pump that bubbles air through air stones every 1 h. The roots and leaves of plants were harvested for RNA extraction. Three biological samples were collected from each tissue. All Arabidopsis thaliana used in this study were in the Columbia ecotype background. The growth conditions of Arabidopsis were set according to our previous work.14 To analyze the expression of stressassociated genes, 4-week-old OE and WT lines were treated with deionized water (control), 300 mM mannitol, or 50 μM ABA for 24 h. Then the rosette leaves were harvested for RNA extraction. Three biological samples were collected from each tissue. Real-Time Quantitative RT-PCR Analysis. FastQuant RT kit (TIANGEN, China) was used to synthesize the first cDNA strand. The reaction system and program of real-time qRT-PCR refer to the protocol described by our previous paper. 1 4 ZmUbi-2 (GRMZM2G419891) and ZmActin1 (GRMZM2G126010) were used as internal controls to study the expression of ZmNAC071. ACT7 (AT5G09810) and TUB2 (AT5G62690) were used as internal controls to analyze the expression of putative target genes in Arabidopsis. All primers used for the real-time qRT-PCR experiments are listed in Table S1. The results are analyzed using the 2−△△ct method.31 Subcellular Localization Assay. The full-length CDS (coding sequence) of ZmNAC071 lacking its termination codon was inserted into the pBI21-GFP vector (primers used are listed in Table S1). The recombinant vectors pBI21-ZmNAC071-GFP and pBI21-GFP (control) were stably transformed into Arabidopsis using the floral dip method. DAPI was used to stain nucleus. The fluorescence signals of the lower epidermis of transgenic plants were visualized by laser scanning confocal microscopy (Leica SP8). Transcriptional Activation Assay. The full and different truncated versions of the CDS of ZmNAC071 were amplified (primers used are listed in Table S1) and inserted into the vector pGBKT7. The different vectors were transformed into yeast strain Y2HGold according to the manufacturer’s protocols (Clontech, USA). The transformants were cultured on SD/-Trp or SD/-Trp/His/-Ade/X-a-Gal medium at 30 °C for 3−5 days. Generation of Arabidopsis Transgenic Plants. The full coding sequence of ZmNAC071 was amplified by PCR (Table S1 for primers used) and inserted into the pROKII vector. The expression of ZmNAC071 was driven by a CaMV 35S promoter. The recombinant vector (35S-ZmNAC071) and empty pROKII vector (35S) were transformed into Arabidopsis using the floral dip method. Homozygous lines were selected on the medium containing kanamycin. ABA and Mannitol Treatments. To study the effects of ABA and mannitol on germination, 1/2MS medium supplemented with 300 mM mannitol or 1.5 μM ABA was used. Germination rates were calculated at sowing for 8 days. For phenotype assays of seedlings, WT, OE lines, and 35S lines seeds were planted in 1/2 MS medium. After 7 days of growth, the germinated seedlings were transferred to 1/2 MS medium with 50 μM ABA or 300 mM mannitol for another 7

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RESULTS Cloning and Sequence Analysis of ZmNAC071. A putative abiotic stress-related NAC gene from maize He344 inbred line was isolated by analyzing transcriptome data (see SRA database with the accession number SRX2672484). It was named ZmNAC071 (GenBank accession no.MH570214) according to blastp results. The open reading frame of ZmNAC071 is 1035bp, encoding a polypeptide of 344 amino acids. Protein sequences alignment showed that ZmNAC071 possesses two domains. The N-terminal NAC domain is relatively conserved between monocotyledon and dicotyledon (Figure S2A). There are five subdomains (A−E) in the NAC

B


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Figure 1. ZmNAC071 gene expression and protein localization. (A) Expression pattern of ZmNAC071 under 300 mM mannitol and 50 μM ABA treatments. Relative expression levels were normalized to 1 in maize He344 plants without stress (0 h). Values are mean (±SD) of three biological replicates. Asterisks denote statistical significance compared to control plants (0 h) using Student’s t test (*, P < 0.05, **, P < 0.01). (B) Subcellular localization of ZmNAC071. Expression of GFP and ZmNAC071-GFP in transgenic Arabidopsis epidermal cells was detected using laser scanning confocal microscopy. GFP, GFP green fluorescence images; DAPI, DAPI for nuclear staining images; Bright, images of brightfield; Merged, the merged images of brightfield, GFP and DAPI staining.

duced into wild-type Arabidopsis, and the positive plants were obtained. We identified the localization using stably transformed Arabidopsis epidermal cells. The Arabidopsis epidermal cells expressing the GFP gene showed green fluorescence signals throughout the cells (Figure 1B). In contrast, the leaf epidermal cells expressing the GFP-ZmNAC071 fusion gene only displayed fluorescence in the nucleuses (Figure 1B). ZmNAC071 Contains a Repression Domain. Previous work showed that the conserved NARD-like (NAC Repression Domain) domain with LVFY motif exists in the N-terminal of some NAC transcriptional repressors.41 Protein sequences analysis displayed that the NARD-like sequences are also present in ZmNAC071 protein (from 106 to 133 amino acid residues, Figure 2A). To determine whether ZmNAC071 is a transcriptional repressor, yeast transcription activity assay was performed. All transformants grew well on the SD/-Trp medium. However, the yeasts containing full-length CDS and the Nterminal NAC domain (amino acids 1−178) of ZmNAC071 could not survive on SD/-Trp/-His/-Ade/X-a-Gal medium (Figure 2B). In contrast, the yeasts harboring the C-terminal region (amino acids 179−344) of ZmNAC071 could grow on the selection medium and display a blue color, indicating that this region has transcriptional activation activity. To further accurately identify the location of the C-terminal activation domain of ZmNAC071, four deletion fragments in the Cterminal region (amino acids 179−344) were inserted into the pGBKT7 vector. The recombinant vectors were transformed into Y2H yeast strains. In contrast with yeasts carrying the fragment with amino acids 179−344, yeasts carrying the

domain. The subdomain C contains a short stretch of basic amino acids (PRDRKYP), which might act as a nuclear localization signal (NLS). Unlike the NAC domain, the Cterminal domain is remarkably divergent (Figure S2A). A phylogenetic tree was further constructed to study the relationship between ZmNAC071 and other NAC proteins from different species. The results showed that ZmNAC071 has higher homology with the NACs from monocotyledons and lower homology with the NACs from dicotyledons (Figure S2B). Although ANAC071 is the nearest homologous gene of ZmNAC071 in Arabidopsis, the identity of the amino acid sequences between ZmNAC071 and ANAC071 is only 33.06% according to multiple sequence alignment by DNAMAN (Figure S2C). ZmNAC071 Gene Expression and Protein Localization. To investigate the expression pattern of ZmNAC071 under mannitol and ABA, three-leaf stage maize seedlings were subjected to the indicated treatments. The expression of ZmNAC071 was highly upregulated by mannitol during the studied period in leaves (Figure 1A). In roots, the expression of ZmNAC071 reached a peak at 3 h but rapidly decreased thereafter under mannitol-induced osmotic stress (Figure 1A). ABA treatments increased ZmNAC071 expression at 0.5−4 h, with a peak at 2 h in leaf and 1 h in root (Figure 1A), which suggested that ZmNAC071 was extremely sensitive to ABA at the early stage of treatments. These results indicate that ZmNAC071 may participate in the ABA and osmotic stress responses in maize. To determine the subcellular localization of ZmNAC071, pBI21-ZmNAC071-GFP and pBI21-GFP vectors were introC


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Figure 2. Transcriptional activation assay of ZmNAC071. (A) Alignment of the NARD-like domain. (B) Analysis of the transcriptional activity of full-length or partial deletion of ZmNAC071 in yeast. Numbers indicate positions of amino acids. Transformants were examined using SD/-Trp (growth control) and SD/-Trp/-His/-Ade/X-a-Gal medium. Transformant carrying empty pGBKT7 plasmid (pBD) was used as a negative control.

weight were detected in four OE lines compared to that in the WT (Figure 3A−E). The 35S plants had generally similar germination rate, root length, and fresh weight to the WT under normal and treatments (Figure 3A−E). In addition, the sensitivity to ABA and mannitol of ZmNAC071-overexpresion lines was further determined at the adult stage in soil. Under normal conditions, the growth performance of OE lines was comparable to that of the WT (Figure 3F). However, OE lines showed more severe osmotic stress symptoms than those of WT (Figure 3F). Forty-six percent of the OE1 lines and 41% of the OE3 lines survived, while 98% of WT survived after 300 mM mannitol treatments for 16 days (Figure 3G). Under the ABA treatments, the OE plants displayed lower chlorophyll content than those of WT lines (Figure 3H). Taken together, these results indicate that ZmNAC071 can improve the sensitivity of plants at various stages in the presence of osmotic stress and ABA. ZmNAC071 Negatively Modulates ROS Scavenging Systems. To investigate whether ZmNAC071 is involved in the regulation of ROS scavenging under osmotic stress and ABA, we compared the levels of two key ROS species H2O2 and O2− in ZmNAC071-overexpression lines and WT lines using DAB and NBT staining, respectively. There is no significant difference in DAB and NBT staining between the WT and the OE lines under normal conditions. However, under mannitol and ABA conditions, the staining in the OE lines was deeper than that in the WT lines in both DAB and NBT assays (Figure 4A and 4B). In addition, the accumulation of H2O2 in guard cells was also measured by H2DCF-DA staining. Similarly, the H2O2 levels in guard cells in OE lines

fragments (amino acids 188−344, 208−344, 218−344, or 302−344) did not grow on the selection medium (Figure 2B). Therefore, the QMSKVSSSSS domain (amino acids 179−188) is crucial for the transactivation activity of the C-terminal region of ZmNAC071. As the full length of ZmNAC071 has no transcriptional activation activity while the C-terminal region (amino acids 179−344) has strong activation ability, we hypothesized that there may be a repression domain in the full-length of ZmNAC071 to inhibit the function of C-terminal activation domain. To test this, four recombinant vectors (134−344, 106−344, 78−344, and 28−344) were also transformed into Y2H yeast strains. The region of 134−344 displayed strong transcriptional activation activity, while the other three versions (106−344, 78−344, and 28−344) exhibited a loss of transactivation activity (Figure 2B). These results suggest that the specific region containing amino acids 106−133 (GLRKTLVFYRGRAPGGERTAWVMHEYRL) can repress the transcriptional activation activity of the C-terminal activation domain of ZmNAC071. ZmNAC071 Negatively Modulates ABA and Osmotic Responses in Plants. The expression levels of seven independent T3 homozygous ZmNAC071-overexpresion lines were determined by real-time qRT-PCR assay (Figure S3). Four independent transgenic lines (OE1, OE3, OE7, OE8) with relatively high transcript levels of ZmNAC071 were used to study gene function. No significant difference in germination rate, root length, and fresh weight was observed between WT and OE lines under normal conditions. However, obvious reductions in germination rate, root length, and fresh D


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Figure 3. Analysis of ABA and osmotic stress tolerance. (A) Germination assay. Seeds of different lines were cultivated on 1/2 MS medium supplemented with mannitol (300 mM) or ABA (1.5 μM) for 8 days. Photographs showed representative seedlings. Bars: 0.5 cm. (B) Germination rates statistical analysis; 100−110 seeds were used per experiment. (C) Root length assay; 7-day-old seedlings were transferred to 1/2 MS medium supplemented with 50 μM ABA or 300 mM mannitol. Photographs were taken 7 days after vertical cultivation. Bars: 1.0 cm. (D, E) Quantification of root length and fresh weight (n = 30). (F) Soil-grown plant phenotypes assay; 4-week-old OE and WT plants were treated with ABA (50 μM) and mannitol (300 mM) for 16 days, and then images were taken. Bars: 1.0 cm. (G) Survival rates statistical analysis. Survival rates were calculated after mannitol (300 mM) treatments for 16 days (n = 100). (H) Chlorophyll contents analysis. Chlorophyll contents were measured after ABA (50 μM) treatments for 16 days. OE: ZmNAC071-overexpression lines; WT: wild type; 35S: Empty pROKII vector control. Values are mean (±SD) of three biological replicates. Asterisks indicate significant differences (*, P < 0.05, **, P < 0.01) between WT and other lines (Student’s t test).

were higher than in WT lines after mannitol and ABA treatments (Figure 4C). Consistent with staining analysis, quantitative results further confirmed that the levels of H2O2 and O2− in ZmNAC071-overexpression lines were significantly higher than that in WT lines after mannitol and ABA treatments (Figure 4D and 4E). These results demonstrate that ZmNAC071 negatively modulates ROS scavenging in the presence of mannitol and ABA. The activities of two key ROS scavenging enzymes SOD (superoxide dismutase) and POD (peroxidase) were further compared in ZmNAC071-overexpression lines and WT lines.

The SOD and POD activities were significantly decreased in OE lines compared to WT plants after mannitol and ABA treatments, although OE and WT lines had no obvious difference in SOD and POD activities under normal conditions (Figure 4F and 4G). In view of POD and SOD activities being affected by ZmNAC071 under mannitol and ABA treatments, we further analyzed the effect of ZmNAC071 on the expression levels of SOD and POD genes. Under normal conditions, except for MSD1, the expression levels of SODs (CSD1/2/3, FSD1/3) and PODs (PRX22/39/69) in the ZmNAC071-overexpression lines were similar to that in WT E


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Figure 4. Detection of ROS accumulation and ROS scavenging capability. (A, B) Analysis of H2O2 and O2− accumulation using DAB and NBT staining, respectively. Bars: 0.5 cm. (C) Analysis of H2O2 accumulation in guard cells using H2DCF-DA staining. (D, E) Quantitative determination of H2O2 and O2− content. (F, G) Measurement of SOD and POD activities. Values are mean (±SD) of three biological replicates. Double asterisks represent significant differences in comparison with WT at P < 0.01 (Student’s t test).

Figure 5. Relative expression levels of SOD and POD genes in different lines after ABA and mannitol treatments. Expression of each gene in WT was set to 1. Values are mean (±SD) of three biological replicates. Asterisks denote significant differences in comparison with WT using Student’s t test (*, P < 0.05, **, P < 0.01).

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Figure 6. Analysis of proline metabolism. (A) Comparison of proline content between WT and OE plants. (B) Relative expression levels of proline biosynthesis and degradation-related genes in WT and OE plants. Expression of each gene in WT was set to 1. Values are mean (±SD) of three biological replicates. Double asterisks represent significant differences in comparison with WT at P < 0.01 (Student’s t test).

Figure 7. Water loss rate and stomatal aperture assays. (A) Water loss rates in detached leaves of the OE lines and WT lines. (B) Image of stomata under laser scanning confocal microscope (Leica SP8). Bars: 5 μm. MES-KCl buffer-mediated stomatal closure was used as a control. (C) Determination of stomatal aperture using the ratio of width to length. At least 30 guard cells were used for each sample. (D) Relative expression of AtMYB61 in WT and OE plants. Expression of AtMYB61 in WT was set to 1. Values are mean (±SD) of three biological replicates. Asterisks denote significant differences in comparison with WT using Student’s t test (*, P < 0.05, **, P < 0.01).

P5CS2) and three proline degradation genes (P5CDH1, P5CDH2, and ProDH) were examined. Under normal growth conditions, the expression levels of these five genes were comparable between the WT and the OE plants. The expression levels of the two proline biosynthesis genes were strongly repressed in the OE lines compared to the WT plants under osmotic stress and ABA. (Figure 6B). In contrast, the expression levels of the three proline degradation genes were highly enhanced in the OE lines compared to WT under osmotic stress and ABA (Figure 6B). Water Loss Rate and Stomatal Aperture Analysis. To study the potential mechanisms of the osmotic stress sensitivity phenotype of ZmNAC071-overexpression lines we measured

lines. However, under osmotic stress and ABA treatments, the SODs (CSD1/2/3, MSD1, FSD1/3) and PODs (PRX22/39/ 69) displayed lower expression levels in the OE plants than that in the WT plants (Figure 5). These results further demonstrate that ZmNAC071 negatively regulates SODs- and PODs-mediated ROS scavenging. ZmNAC071 Affects Proline Metabolism. There is no obvious difference in the proline levels between WT and ZmNAC071-overexpression lines under normal conditions. However, when exposed to mannitol and ABA conditions, the proline levels were significantly lower in ZmNAC071 transgenic plants compared to the WT (Figure 6A). Next, the expression of two proline biosynthesis genes (P5CS1 and G


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Figure 8. Relative expression levels of ABA and stress-related genes in OE and WT lines under ABA and mannitol treatments. Expression of each gene in WT was set to 1. Values are mean (±SD) of three biological replicates. Double asterisks represent significant differences in comparison with WT at P < 0.01 (Student’s t test).

water loss rate and stomatal aperture. The ZmNAC071overexpression lines showed increased water loss compared to WT (Figure 7A). The stomatal apertures were similar between ZmNAC071-overexpression lines and WT under normal conditions. However, OE1 and OE3 displayed significantly larger stomatal openings compared to WT after mannitol and ABA treatments (Figure 7B and 7C), indicating that ZmNAC071 negatively affects stomatal movement under adverse environmental conditions. It is known that AtMYB61 (AT1G09540) functions as an aperture-regulation gene in Arabidopsis. AtMYB61-overexpression lines showed enhanced tolerance to drought via reducing the stomatal aperture.42 Some abiotic stress-related TFs control the opening of stomatal aperture by regulating AtMYB61 or its homologous genes.38,43,44 To investigate whether the overexpression of ZmNAC071 in Arabidopsis affects the expression of AtMYB61, the real-time qRT-PCR analysis was performed. Under normal conditions, the expression of AtMYB61 in OE lines was comparable with that in WT. Nevertheless, the expression of AtMYB61 was significantly reduced in the ZmNAC071-overexpression lines under mannitol and ABA treatments (Figure 7D), suggesting that ZmNAC071 negatively regulates the expression of AtMYB61 gene under these conditions. ZmNAC071 Inhibits ABA and Stress-Related Genes. To probe other possible regulatory mechanisms of ZmNAC071-mediated abiotic stress response, the expression levels of three ABA-responsive genes ABI3, ABI5, and RD29A and five stress-responsive genes LEA3, LEA7, LEA14, AtDREB1A, and AtDERB2A were analyzed in ZmNAC071 transgenic plants and WT by real-time qRT-PCR. As shown in Figure 8, the expression levels of these eight genes were not significantly changed under normal growth conditions. However, when exposed to osmotic stress or ABA treatments, the expression levels of these genes were highly reduced in

ZmNAC071-overexpression lines compared to WT lines (Figure 8). ZmNAC071 Binds to the CGT[G/A] Core Sequences. To examine whether ZmNAC071 can recognize the motifs from different species, one copy of 5 known NACRS (R1−5) as well as core binding site (R6) and mutated core site (R7) were fused to pHIS2 (Figure 9A). The bindings of ZmNAC071 to these motifs were analyzed using a Yeast one Hybrid (Y1H). All cotransformants could grow normally on the DDO medium. The yeast cells carrying p53HIS2/ pGADT7-p53 (positive control) and pHIS2-R1-6/pGADT7ZmNAC071 could grow well on the TDO medium with 60 or 100 mM 3-AT, while pHIS2-R7/pGADT7-ZmNAC071 and p53HIS2/pGADT7-ZmNAC071 (negative control) could not grow normally on selection medium (Figure 9A). These results demonstrate that ZmNAC071 interacts with the known 5 motifs (R1−5), and the core sequence CACG/CGTG or CGTA/TACG (abbreviated as CGT[G/A]) is crucial for ZmNAC071 protein recognition. To study the distribution of core sequences CGT[G/A] we analyzed the promoters regions (from −1500 to −1) of 20 putative target genes that were significantly downregulated by ZmNAC071. It was found that all of the promoters of putative target genes contain at least four or more CGT[G/A] (Table S2). Thus, it seems that CGT[G/A] is essential for ZmNAC071 to regulate its target genes. To verify ZmNAC071 can bind directly to putative target genes promoters containing CGT[G/A] core sites, the promoter fragments of MSD1, PRX22, DREB2A, AtMYB61, P5CS2, ABI3, LEA7, and LEA14 were isolated via genomic PCR. The schematic diagrams of the promoter regions of these genes are shown in Figure S1. The bindings of ZmNAC071 to these promoter fragments were investigated in yeast. We found that all of the cotransformants except negative control showed binding affinity on the selection medium (Figure 9B). These results suggest that ZmNAC071 can directly interact with the H


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Figure 9. Bindings of ZmNAC071 to NACRS and the promoters of target genes. (A) Y1H analyses of the interactions between ZmNAC071 and NACRS containing CGT[G/A] core sequences. pHIS2-R (1−7): One copy of five known NACRS (R1−5), one core binding site (R6), and one mutant type (R7) were fused to the pHIS2 as the reporter vectors. pGADT7-ZmNAC071: CDS of the ZmNAC071 was inserted into pGADT7Rec2 as effector vector. (B) Determination of the interactions between ZmNAC071 and the target genes promoters in yeast. Promoters containing at least 2 core sequences were fused to the pHIS2 vector as reporter constructs. Cotransformants of the effector vector with each reporter vector were analyzed by cultivating serial dilutions (1, 10−1, 10−2 and 10−3) of yeast on TDO medium with 3-AT. p53HIS2/pGADT7-p53: Positive controls. p53HIS2/pGADT7-ZmNAC071: Negative controls.

stress responses.12,13 Here, we isolated a NAC transcriptional repressor gene ZmNAC071 from maize. The previous study has displayed that GmNAC20, GmNAC11, AtNAC2, NST1, ATAF1, RD26, and SNAC protein shared conserved NAC transcription repression domain (NARD-like domain) in their N-terminal regions, and the motif LVFY in NARD-like domain was considered to be of importance for transcriptional repression activity.41 The relative conserved NARD-like region with the LVFY motif also exists in ZmNAC071 protein (from amino acid 106 to 133: GLRKTLVFYRGRAPGGERTAWVM-

promoter fragments of MSD1, PRX22, DREB2A, AtMYB61, P5CS2, ABI3, LEA7 and LEA14 containing core sequences CGT[G/A].

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DISCUSSION With the identification of numerous NAC-type transcriptional activators, the activation mechanism of abiotic stress responses mediated by NAC proteins is relatively well studied.26,27,45−47 Recent studies showed that some NAC-type transcriptional repressors could also participate in the regulation of abiotic I


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drought tolerance by decreasing the stomatal aperture.42 Some abiotic stress-related TFs could positively regulate AtMYB61 or its homologous genes to reduce the stomatal aperture under abiotic stress conditions. For example, AST1 induces stomatal closure by positively regulating AtMYB61 expression, thereby aiding abiotic stress tolerance.43 Similarly, overexpressing BplMYB46 in Betula platyphylla reduces the stomatal apertures via upregulating the expression of BpMYB61, leading to decreased water loss rates.38 Contrary to these results, ZmNAC071 could significantly increase the stomatal apertures by downregulating the expression of AtMYB61 under ABA and mannitol (Figure 7D), thus resulting in the increased water loss. TFs regulate the expression of a series of genes through binding to cis-acting elements that existed in the promoter of these genes during plants in response to abiotic stress. Thus, identification of corresponding cis-acting elements of each TF will help us better understand the underlying regulatory mechanisms. Our previous work showed that Arabidopsis ANAC069 protein mainly binds to C[A/G]CG[T/G] sequences to downregulate the expression of genes under abiotic stress conditions.14 In the present work we found that ZmNAC071 bound to CGT[G/A] core sequences specifically (Figure 9A), and these core sequences broadly existed in the promoters of stress-related genes downregulated by ZmNAC071 (Table S2). The core sequence CGT[G/A] is similar to the reverse complementary sequences of ANAC069 target cis-element C[A/G]CG[T/G].14 Besides, ZmNAC071 could also directly interact with the promoter fragments containing CGT[G/A] core sequences (Figure S1 and Figure 9B). These results confirmed that CGT[G/A] core sequences are important for gene expression regulation mediated by ZmNAC071. Consistently, Hu et al. also found that BpNAC012 from Betula platyphylla regulated the expression of stress-related genes via recognition of the CGT[G/A] core sequences within their promoter.28 Taken together, we believe that NAC proteins from different species can recognize the similar core sequences CGT[G/A] under abiotic stress conditions. The NAC-specific core sequences CGT[G/A] are also enriched in the promoter regions of DREB1A/2A, RD29A, and LEA3/7/14 (Table S2), and these six genes were downregulated in ZmNAC071-overexpression plants under ABA and osmotic stress conditions (Figure 8). DREB1A (CBF3) and DREB2A participate in the regulation of abiotic stress responses through affecting the expression of a series of target genes.54−56 RD29A encodes a low-molecular-weight hydrophilic protein, which helps to retain water under adverse circumstances.57 LEA proteins are beneficial to maintain the stability of cell membrane and to retard irreversible protein aggregation under adverse conditions.58 Coincidentally, both RD29A and LEA14 are known as direct target genes of DREB2A,55 which suggests that ZmNAC071 may downregulate the expressions of RD29A or LEA14 in direct and indirect two ways under ABA and osmotic stress. For the direct way, ZmNAC071 directly binds to the promoter region of RD29A or LEA14 with CGT[G/A] core sequences to downregulate their expression. For the indirect way, ZmNAC071 first downregulates the expression of DREB2A gene via binding to CGT[G/A] in its promoter, and then reduced DREB2A protein will lead to decreased expression levels of RD29A and LEA14. Altogether, decreased expression of these stress-responsive genes in ZmNAC071 transgenic

HEYRL) (Figure 2A). The C-terminal region (179−344) of ZmNAC071 and extended C-terminal region (134−344) without the NARD-like region have strong transcriptional activation activity. However, the full length of ZmNAC071 and the extended C-terminal regions containing the NARD-like region (106−344, 78−344, and 28−344) have no transcriptional activation activities (Figure 2B). Since the NARD-like region can repress the transcriptional activation activity of the C-terminal activation region, the transcriptional activation ability of the full-length ZmNAC071 protein is lost. Hao et al. found that the ultimate function of NAC TFs containing both transcriptional activation domain and repression domain depends on the interaction between these two domains.41 Thus, the reason why ZmNAC071 acts as a transcriptional repressor may be that the NARD-like domain in the Nterminal region is more powerful than the C-terminal transcriptional activation domain. The roles of NAC TFs in regulation of the antioxidant system during plants responding to abiotic stresses have been well studied in many species, but limited studies exist in maize.14,26−28,48,22 In this work, ROS accumulated to the higher levels in ZmNAC071-overexpression plants than that in WT plants under mannitol and ABA (Figure 4A−E). Besides, ZmNAC071 expression levels are negatively correlating with POD and SOD activities under mannitol and ABA treatments (Figure 4F and 4G) and also negatively correlating with the expression levels of many SODs and PODs (Figure 5). Therefore, ZmNAC071 functions as a negative regulator of ABA and osmotic stress by controlling the expression of downstream genes involved in the ROS pathway, leading to decreased SOD and POD activities and increased ROS accumulation. The high levels of ROS can cause membrane lipid peroxidation, leading to a large accumulation of MDA.49 Consistent with this, ZmNAC071-overexpression plants displayed more severe cell membrane damage and higher MDA levels compared to WT under ABA and osmotic stress (Figure S4). Altogether, we believe that ZmNAC071 from maize can also participate in the regulation of the antioxidant system under adverse environmental conditions. Proline can be used as an index to evaluate plant abiotic stress tolerance.50,51 There are five proline metabolism-related genes in Arabidopsis, including two proline biosynthesis genes (P5CS1 and P5CS2) and three proline degradation genes (P5CDH1, P5CDH2, and ProDH).52,53 In this work, the expression levels of P5CS1 and P5CS2 were lower in OE lines compared to WT, while the expression levels of P5CDH1, P5CDH2, and ProDH were higher in OE lines compared to WT under osmotic stress and ABA (Figure 6B). Correspondingly, proline levels in OE lines were lower than that in WT under osmotic stress and ABA (Figure 6A). These results suggest that overexpressing ZmNAC071 reduces the proline content via inhibiting proline biosynthetic genes and inducing proline degradation genes, leading to enhanced sensitivity of transgenic lines to ABA and mannitol-induced osmotic stress. The transpiration rate is closely related to the stomatal opening. Our results showed that overexpressing ZmNAC071 in Arabidopsis can improve the transpiration rate (Figure 7A). Therefore, we compared the stomatal apertures between ZmNAC071-overexpression lines and WT. The stomatal apertures in OE lines were dramatically larger compared to WT under ABA and osmotic stress (Figure 7B and 7C). AtMYB61 is the first TF involved in the regulation of stomata closure. AtMYB61 transgenic Arabidopsis showed enhanced J


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Figure 10. Working model of ZmNAC071 under osmotic stress and ABA. ABA or osmotic stress induces the expression of ZmNAC071. Activated ZmNAC071 inhibits the ABA/stress-associated genes by binding to CGT[G/A] core sequences, which results in reduced ROS scavenging capability and proline content, increased water loss rate, and membrane damage. These physiological changes finally improve the sensitivity of ZmNAC071 transgenic plants to ABA and osmotic stress.

function as positive regulators in ABA-associated abiotic stress responses.57,63,64 The reduced expression levels of ABI3, ABI5, and RD29A in ZmNAC071-overexpression lines lead to enhanced sensitivity to ABA and osmotic stress. In conclusion, we demonstrated that the expression of ZmNAC071 in the maize inbred line He344 was induced by osmotic stress and ABA. ZmNAC071 was confirmed as a transcriptional repressor due to subcellular localization in the nucleus, the presence of transcriptional inhibitory activity, and binding to core sequences CGT[G/A] to downregulate its target genes. Overexpressing ZmNAC071 in Arabidopsis significantly enhanced sensitivity to osmotic stress. The enhanced sensitivity is ascribed to increased ROS level, stomatal aperture, water loss rate, and membrane damage as well as decreased ROS scavenging capability, proline content, and membrane stability (Figure 10). Besides, another possible reason is due to altered expression of an array of stress and ABA-responsive genes in ZmNAC071-overexpression plants (Figure 10). These strategies may act in cooperation to render a more sensitive line of defense against the ABA and osmotic stress.

plants led to reduced endurance to abiotic stress, which further supports that ZmNAC071 is a negative regulator. Further investigations are needed to identify the cis-elements and downstream genes targeted by ZmNAC071 in maize. ABA can act as a mediator to trigger a series of responses in part of stress-signaling pathways.59 NAC TFs participate in regulating stress responses through ABA-dependent or ABAindependent signaling pathways.10,11,27,60−62 ZmNAC071 is likely to function as a negative regulator via the ABAdependent pathway under abiotic stress conditions. There are three reasons to support our hypothesis. First, the expression of ZmNAC071 is dramatically induced by exogenous ABA application (Figure 1A). Second, ZmNAC071 overexpression conferred hypersensitivity to ABA at various stages. The germination rates were significantly lower in ZmNAC071 transgenic plants compared to the WT under ABA treatments (Figure 3A and 3B). At seedling stages, root length and fresh weight in OE plants were more severely inhibited by ABA in comparison with WT (Figure 3C−E). Furthermore, ZmNAC071-overexpression lines at the adult stages showed lower chlorophyll and proline content, and SOD and POD activities than those of wild type after ABA treatments (Figure 3H; Figure 6A; Figure 4F and 4G). Third, the expression levels of some ABA signal transduction pathway genes such as ABI3, ABI5, and RD29A were significantly downregulated by the overexpression of ZmNAC071 under both ABA and osmotic stress (Figure 8). RD29A, ABI3, and ABI5 were reported to

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02331. K


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Primers used in this study; distribution of the CGT[G/ A] core sequences in the promoter regions of genes regulated by ZmNAC071; schematic diagrams of promoter regions of ZmNAC071 downstream abiotic stress-responsive genes used for Y1H analysis; sequence analysis of ZmNAC071; relative expression of ZmNAC071 in the overexpressing lines; ZmNAC071 modulates membrane damage (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: 86-13836961072. *E-mail: [email protected]. Tel.: 86-13936756001. ORCID

Lin He: 0000-0002-5737-1019 Kejun Yang: 0000-0002-4654-8042 Author Contributions †

L.H. and J.B.: These authors contributed equally to this work.

Author Contributions

L.H. and K.Y. conceived the experiment. L.H. and J.B. performed experimental work and analyzed the data. L.H. and K.Y. provided financial support. L.H. wrote the article. J.X. modified the language of the paper. All authors read and approved the final version of the manuscript. Funding

This work was financially supported by the National Natural Science Foundation of China (No. 31701328), Postdoctoral Science Foundation of Heilongjiang Province (LBH-Z16165), and Program for Young Scholars with Creative Talents in Heilongjiang Bayi Agricultural University (No. YJSCX-2017Y01). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED NAC, NAM, ATAF1/2, and CUC2; TF, transcription factor; ORF, open reading frame; qRT-PCR, quantitative real-time polymerase chain reaction; GFP, green fluorescence protein; CDS, coding sequence; ABA, abscisic acid; NACRS, NAC recognition sequence; DAPI, 4′,6-diamidino-2-phenylindole; DAB, 3,3′-diaminobenzidine; NBT, nitroblue tetrazolium; H2DCF-DA, 2,7-dichlorofluorescin diacetate; PI, propidium iodide; 3-AT, 3-amino-1,2,4-triazole; Y1H, Yeast one-Hybrid; Y2H, Yeast two-Hybrid

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Novel Maize NAC Transcriptional Repressor ZmNAC071 Confers

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