Plant Cell Rep (2014) 33:767–778 DOI 10.1007/s00299-014-1602-y
ORIGINAL PAPER
A novel NAC transcription factor from Suaeda liaotungensis K. enhanced transgenic Arabidopsis drought, salt, and cold stress tolerance Xiao-lan Li • Xing Yang • Yu-xin Hu Xiao-dong Yu • Qiu-li Li
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Received: 3 January 2014 / Revised: 28 February 2014 / Accepted: 12 March 2014 / Published online: 29 March 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Key message SlNAC1 functions as a stress-responsive NAC protein involved in the abscisic acid-dependent signaling pathway and enhances transgenic Arabidopsis drought, salt, and cold stress tolerance. Abstract NAC (NAM, ATAF1, 2, CUC2) transcription factors constitute the largest families of plant-specific transcription factors, known to be involved in various growth or developmental processes and in regulation of response to environmental stresses. However, only little information regarding stress-related NAC genes is available in Suaeda liaotungensis K. In this study, we cloned a full-length NAC gene (1,011 bp) named SlNAC1 using polymerase chain reaction from Suaeda liaotungensis K. and investigated its function by overexpression in transgenic Arabidopsis. SlNAC1 contains an NAC-conserved Communicated by Q. Zhao.
Electronic supplementary material The online version of this article (doi:10.1007/s00299-014-1602-y) contains supplementary material, which is available to authorized users. X. Li X. Yang Y. Hu X. Yu Q. Li (&) College of Life Sciences, Liaoning Normal University; Key Laboratory of Plant Biotechnology in Liaoning Province, 1 South Liushu Street, Ganjingzi District, Dalian 116081, Liaoning, China e-mail: [email protected] X. Li e-mail: [email protected] X. Yang e-mail: [email protected] Y. Hu e-mail: [email protected] X. Yu e-mail: [email protected]
domain. Its expression in S. liaotungensis was induced by drought, high-salt, and cold (4 °C) stresses and by abscisic acid. Subcellular localization experiments in onion epidermal cells indicated that SlNAC1 is localized in the nucleus. Yeast one-hybrid assays showed that SlNAC1 functions as a transcriptional activator. SlNAC1 transgenic Arabidopsis displayed a higher survival ratio and lower rate of water loss under drought stress; a higher germination ratio, higher survival ratio, and lower root inhibition rate under salt stress; a higher survival ratio under cold stress; and a lower germination ratio and root inhibition rate under abscisic acid treatment, compared with wildtype Arabidopsis. These results suggested that SlNAC1 functions as a stress-responsive NAC protein involved in the abscisic acid-dependent signaling pathway and may have potential applications in transgenic breeding to enhance crops’ abiotic stress tolerances. Keywords Abiotic stress NAC transcription factor Transgenic plant Stress resistance Suaeda liaotungensis K. Abbreviations ABA Abscisic acid CBF Core binding factor DREB Dehydration response element binding factor GFP Green fluorescent protein MYB Myeloblastosis NAC NAM (no apical meristem), ATAF1 or ATAF2 and CUC2 (cup-shaped cotyledon) ORF Open reading frame PEG Polyethylene glycol 6000 6000 RT-qPCR Real-time quantitative polymerase chain reaction
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SNAC TFs
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Stress-responsive NAC Transcription factors
Introduction Environment stresses, such as drought, high salinity, and cold, reduce productivity and cause significant crop losses globally. Drought and salinity affect more than 10 % of arable land, which results in more than 50 % decline in the average yields of important crops worldwide (Dracup et al. 1998). Tolerance or susceptibility to these stresses is a complex process, as stress may affect multiple stages of plant development and often several stresses concurrently affect the plants (Chinnusamy et al. 2004). Therefore, plants have evolved a series of mechanisms to survive such environmental adversities. Plant stress tolerance involves changes at whole-plant, tissue, cellular, physiological, and molecular levels. Molecular responses to abiotic stress include perception, signal transduction, gene expression and, ultimately, metabolic changes in the plant, resulting in stress tolerance (Agarwal et al. 2006). Transcription factors (TFs) and their corresponding cis-regulatory sequences act as molecular switches for gene expression, regulating their temporal and spatial expression. Increasing evidence demonstrates that numerous transcription factors, such as DREB, CBF, bZIP, zinc finger, MYB, and NAC, are directly or indirectly involved in the regulation of plant defense and stress responses (Thomashow 1999; Zhu 2002; Seki et al. 2003; Shinozaki et al. 2003; Fujita et al. 2004; Mukhopadhyay et al. 2004; Yanhui et al. 2006). NAC (NAM, ATAF1, 2, CUC2) transcription factors constitute the largest families of plant-specific transcription factors and the family is present in a wide range of land plants. NAC was derived from the names of the first three described TFs containing NAC domain, namely NAM (no apical meristem), ATAF1-2, and CUC2 (cup-shaped cotyledon) (Souer et al. 1996; Aida et al. 1997). The NAC TFs are associated with diverse processes, which include various developmental programs, senescence, formation of secondary walls, and biotic and abiotic stress response (Puranik et al. 2012). Stress-inducible NAC genes have been shown to be involved in abiotic stress tolerance (Nakashima et al. 2011). Transgenic plant overexpressing stress-responsive NAC (SNAC) genes have exhibited at least one type of improved abiotic stress tolerance (Nakashima et al. 2011). Arabidopsis ATAF1, ANAC019, ANAC055, and ANAC072 were found to be induced by drought, high salinity, and abscisic acid (ABA), and their
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overexpression in transgenic plants showed remarkably enhanced plant tolerance to drought (Wu et al. 2009; Mauch-Mani and Flors 2009; Jensen et al. 2009; Tran et al. 2004). Rice ONAC045, OsNAC5, OsNAP, SNAC1, wheat TaNAC67, and maize ZmSNAC1 were reported to be drought, high-salt, and low-temperature stresses, and abscisic acid (ABA)-inducible genes. Plants individually overexpressing these genes showed an increase in drought tolerance or salt tolerance, or low temperature tolerance (Zheng et al. 2009; Takasaki et al. 2010; Chen et al. 2014; Hu et al. 2006; Liu et al. 2014; Mao et al. 2014; Lu et al. 2012). These studies indicate that SNAC transcription factors have important roles for the control of abiotic stress tolerance and that their overexpression can improve transgenic plants stress tolerance. However, there is no information on the abiotic stressrelated NAC proteins in Suaeda liaotungensis K, a halophytic plant, which grows widely by the seashore in Dalian, China, as a source of genetic material for introduction into non-salt-tolerant crops. We discovered a sequence annotated as an NAC transcription factor from the database of S. liaotungensis transcriptome sequencing and named this protein SlNAC1. To identify whether SlNAC1 was related to abiotic stress, we designed preliminary tests and found that SlNAC1 was induced by salt stress. In this study, we cloned SlNAC1 and characterized its function. A search for the conserved domains in NCBI showed that SlNAC1 contained an NAC domain. The expression of SlNAC1 was induced by drought, high salt, cold, and abscisic acid (ABA), as assessed by real-time quantitative polymerase chain reaction. Its overexpression improved the drought, high-salt, and cold stress tolerance of transgenic plants. Furthermore, SlNAC1 is localized in the nucleus and functions as a transcriptional activator, as determined by subcellular localization and transactivation assays. Therefore, SlNAC1 may become a hot topic for abiotic stress resistance research and is expected to be used in genetically modified crops.
Materials and methods Plant materials Green leaves and seeds of wild S. liaotungensis were collected from the seashore in Dalian. The leaves were used for SlNAC1 cDNA cloning. The seeds were used to breed seedlings for gene expression analysis. Arabidopsis thaliana accession Columbia (Col-0) was used for gene transformation and as a phenotypic control and was grown in a controlled environment chamber at 22 °C, with a 16/8 photoperiod, light intensity of 120–150 lmol m-2 s-1, and 60 % relative humidity.
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SlNAC1 isolation and sequence analysis The sequence of SlNAC1 (GenBank accession number KC176074) was obtained from the database of S. liaotungensis transcriptome sequencing. It was 1,011-bp long and contained a full-length open reading frame (ORF). To obtain the ORF by the reverse transcription polymerase chain reaction (RT-PCR) approach, two primers were used (sense: 50 -CTCTCTATCTCACCTAAAGTTTG C-30 and antisense: 50 -GGAAGTCAATCATGGAATCTCTAC-30 ). The PCR conditions for amplifying SlNAC1 were as follows: 3 min pre-denaturation at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 50 °C, 1 min at 72 °C; and a final extension for 10 min at 72 °C. The PCR product was purified and cloned into the pEASY-T1 cloning vector (TRANSGEN BIOTECH, China) for sequencing (Takara, Japan). Alignment of SlNAC1 and other NAC proteins was performed using the ClustalX program (version 1.83) and viewed by GeneDoc software (version 2.5). A phylogenetic tree was constructed using the MEGA program (version 4.0) by the neighbor-joining method. The parameters of pairwise deletion and the p-distance model were used. A bootstrap test of phylogeny was performed with 1,000 replicates.
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was conducted on a Thermal Cycler Dice Real Time System TP800 (Takara, Japan) using SYBR Premix Ex Taq II (Tli RnaseH Plus) (Takara, Japan). Baseline and threshold cycles (Ct) were automatically determined using the Thermal Cycler Dice Real Time System TP800 Software release Version 3.0. Relative gene expression with respect to SlActin was determined as described previously, using the 2 (-DD CT) method (Livak and Schmittgen 2001). Subcellular localization of SlNAC1 The full-length ORF of SlNAC1 was amplified with the primers 50 -gcGAATTCATGGGAGCTTTAATTATAA GAGA A-30 and 50 -gcGGATCCCTACCAATCATGT CTTGTATT GC-30 , digested with EcoRI and BamHI, and fused to the 30 end of GFP in the pEGAD vector to generate the fusion construct 35S::GFP-SlNAC1. The fusion gene and the negative control pEGAD vector were transformed into living onion epidermal cells by biolistic bombardment with a GeneGun (GJ-1000; China), according to the instruction manual (helium pressure, 9 MPa). Protein expression was observed under a confocal laser-scanning microscope (Ti-E; Nikon). Transactivation assay of SlNAC1
Expression analysis of SlNAC1 under different treatments Seedlings (4 weeks old) of S. liaotungensis were used for the various stress treatments. The treatments were executed at 25 °C and 50–65 % humidity and the seedlings were grown in 1/10 MS culture medium with 200 mM NaCl, 100 lM ABA, and 10 % PEG 6000 (polyethylene glycol 6000), respectively. For cold treatment, seedlings were transferred to a growth chamber at a temperature of 4 °C. For gene expression analysis, total RNA was isolated from liquid nitrogen-frozen seedlings using RNAiso Plus reagent (Takara, Japan), according to the manufacturer’s protocol. RNA integrity was verified by 1 % agar gel electrophoresis. The first-strand cDNA was synthesized with 1 lg of total RNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan), according to the manufacturer’s procedure. Primers were designed using Beacon Designer version 7.0 (Premier Biosoft international, Palo Alto, CA, USA) with melting temperatures of 60–65 °C, primer lengths of 20–24 bp, and amplicon lengths of 90–200 bp. The primers of SlNAC1 and the internal reference gene SlActin (a S. liaotungensis actin gene, GenBank accession number: JX860282.1) used for qRT-PCR were 50 -AGCGTACTCACTGGGTTATCCAT30 and 50 -ATCGGCAAAGAACAAAAGCAC-30 , and 50 ATCCCAAGGCTAATCGTGAAAA-30 and 50 -CAC 0 CATCACCAGAGTCCAACA-3 , respectively. qRT-PCR
The entire coding region of SlNAC1 was fused to the GAL4 DNA-binding domain in the pGBKT7 vector. According to the manufacturer’s protocol (Stratagene, USA), pGBKT7SlNAC1 and the negative control pGBKT7 vector were transferred into the yeast strain AH109, respectively. The transformed yeast cells were grown on SD glucose medium lacking Trp, and medium lacking Trp, His, and Ade, respectively. The transactivation activity of each protein was evaluated according to its growth status and b-galactosidase filter lift assay (Yeast Protocols Handbook; Clontech, USA). Abiotic stress assays of transgenic Arabidopsis plants The transformation vectors harboring the 35S::GFP or 35S::GFP-SlNAC1 constructs were introduced into Agrobacterium tumefaciens (GV3101) and then transferred into wild-type (Wt) Arabidopsis by the floral dip transformation method (Clough and Bent 1998). Positive transgenic lines of T1 and T2 generations were screened on MS agar medium containing 37.5 lM Basta. The T2 generation was selected according to its segregation ratio (resistant:sensitive = 3:1), and confirmed by genomic PCR. The selected transgenic lines (pEGAD-SlNAC1-1 and pEGADSlNAC1-2, N1 and N2) that displayed the segregation ratio were chosen for further analysis. Expression levels of SlNAC1 were determined by semi-quantitative RT-PCR.
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Total RNA samples were extracted from appropriate plant materials using the RNAiso Plus kit (Takara, Japan). Synthesis of first cDNA, RT-PCR was performed as described above. Expression levels were quantitated by agar gel electrophoresis. T2 generation seeds were sterilized, suspended in 0.1 % agar, and plated on MS medium (including 37.5 lM Basta), then stratified in darkness at 4 °C for 2 days and transferred to growth chambers with the same environmental conditions as described above. Ten-day-old seedlings were transferred from MS plates to water-saturated soil for 7 days, water was withheld from one group until the plants showed evident drought-stressed phenotypes and then the plants were re-watered for drought stress treatment. A second group was watered with 200 mM NaCl solution until the plants showed evident salt-stressed phenotypes for high salt stress treatment. A third group was grown for another 2 weeks, was treated at -20 °C for 90 min, and transferred to normal conditions. For the leaf water loss assay, rosette leaves excised from plants grown in soil were placed on filter paper for air-drying treatment. The leaf weight was measured over a series of time points. To conduct the root growth assay, seeds sown on MS plates were stratified for 2 days at 4 °C and grown vertically for 3 days under normal conditions, before the seedlings were transferred to vertical square MS plates with ABA (10 lM) or NaCl (200 mM). To conduct the germination rate assay, seeds of transgenic lines (N1 and N2) and control lines (wild-type and vector control) were placed on MS medium without or with ABA (10 lM), NaCl (200 mM) and 37.5 lM Basta, respectively. Seeds were vernalized at 4 °C in the dark for 3 days and then transferred to growth chambers. The germination ratio was calculated on the sixth day. Statistical analysis For qRT-PCR, the 2 -DD CT method was used to calculate relative expression levels of the target gene in stressed and non-stressed leaves and other tissues. Significant differences in relative expression levels were identified using a one-way analysis of variance test (P = 0.05; n = 3) using SPSS 13.0 (SPSS Inc., USA).
Results
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presence of the NAC domain in SlNAC1 (Fig. 1a). Phylogenetic analysis revealed that SlNAC1 was clustered into the TIP subgroup of Subfamily I (Fig. 1b). The proteins used in the alignment and phylogenetic tree all had an NAC domain and were obtained by database searching in NCBI. SlNAC1 is a potential stress-related gene To identify whether SlNAC1 could be induced by abiotic stresses, the expression pattern of SlNAC1 in different tissues of wild S. liaotungensis and under various abiotic stress treatments in young leaves was investigated using RT-qPCR. The results showed that the expression of SlNAC1 was the highest in the leaves (Fig. 2a) and that SlNAC1 was induced by drought (10 % PEG 6000) (Fig. 2b), high salt (200 mM NaCl) (Fig. 2c), cold (4 °C) (Fig. 2d), and 100 lM ABA (Fig. 2e). The expression patterns and maximum expression levels differed for each stress treatment. The relative expression levels peaked at 0.5 h for drought, 0.5 h for NaCl, 3 h for cold, and 1 h for ABA, with the corresponding maxima being 2.82-, 9.28-, 6.28-, and 1.92-fold greater than the control (0 h), respectively. SlNAC1 functions as a transcriptional activator and is localized in the nucleus To determine the subcellular localization of SlNAC1, a GFP-SlNAC1 fusion construct and the GFP control in pEGAD driven by the CaMV 35S promoter were transiently expressed in onion epidermal cells and analyzed by confocal laser-scanning microscopy. Figure 3a–c shows that the GFP-SlNAC1 fusion protein was targeted to the nuclei of the cells, while the control GFP protein was located in the cytoplasm (Fig. 3d–f). This result indicated that SlNAC1 was a nuclear protein. The yeast one-hybrid system was used to identify a possible transcriptional activation function of SlNAC1. As shown in Fig. 4, all transformants grew well on SD/Trpmedium. However, only transformants containing pGBKT7-SlNAC1 could grow on SD/Trp-/His-/Ade-medium and showed b-galactosidase activity that cells containing pGBKT7 could not (Fig. 4). These results indicated that SlNAC1 functioned as a transcriptional activator. Overexpression of SlNAC1 confers drought tolerance on transgenic Arabidopsis
SlNAC1 encodes a protein with an NAC domain The SlNAC1 gene encodes a 336-amino acid protein with a conserved NAC domain containing five subdomains at its N terminus (supplemental data 1). Multiple alignments with NAC proteins from other species confirmed the
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We showed that SlNAC1 is a drought-inducible gene (Fig. 2b); therefore it is likely to regulate drought signaling. To characterize such a function, a whole-plant drought assay was performed in soil using T2 generation transgenic lines to test SlNAC1 expression levels (Fig. 5a). Seedlings
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Fig. 1 Multiple alignment of SlNAC1 and phylogenetic tree analysis. a Multiple alignments of NAC proteins from TIP subgroup. Identical amino acid residues are highlighted in red. The regions annotated indicate the subdomains of NAC domain. b Phylogenetic tree of NAC proteins from different species. SlNAC1 was labeled with a big red dot. All of the proteins used in the phylogenetic tree came from database of NCBI. The corresponding accession numbers of the names are as follows: CaNAC6, gi|242877185|; AtNAC36, gi|145360022|; NtTERN, gi|4996349|; AtNTM1, gi|145332955|; ANAC001, gi|297337931|; AtNAC089, gi|75165521|; AtNAC60, gi|332644364|; HvTPA, gi|371925001|; OsNAC8, gi|6730946|; ZmTPA, gi|414876898|; AtVND1, gi|15227958|; OsNAC7, gi|6730944|; AtNAC014, gi|42571727|; AtNAC62, gi|15229161|;
AtTIP, gi|30689531|; MtNAC969, gi|359360012|; AtNAP, gi|18409291|; AtNAC2, gi|6456751|; AtSENU5,gi|9955562|; AhNAC2, gi|209171097|; AhNAC3, gi|209171095|; AtRD26, gi|18416983|; AtNAC019, gi|15219112|; AtNAC055, gi|15232604|; ATAF1, gi|15223456|; GmNAC2, gi|66394512|; AmNAC1, gi|154362215|; SsNAC23, gi|58013003|; ZmNAC1, gi|165855636|; SNAC2, gi|311815437|; TaNAC3, gi|296044564|; SNAC1, gi|88770831|; OsNAC3, gi|6730936|; Ta296044566, gi|296044566|; TaNAC7, gi|292659254|; AtNAC1, gi|6649236|; Zm413953607, gi|413953607|; ANAC011, gi|297336836|; AtCUC1, gi|15232511|; AtNAM, gi|6456751|; TaNAC8, gi|292659256|; TaNAM, gi|292659395|; TaNTL5, gi|292659258| (color figure online)
(10 days old) were transferred from MS plates to watersaturated soil for 7 days, and then water was withheld to cause a severe water deficit in soil. After 21 days, most of the Wt, pEGAD [vector control (V)], and pEGAD-SlNAC1 (N1, N2) plants had wilted because of the extreme water
deprivation (Fig. 5b). After re-watering, 62–76 % of pEGAD-SlNAC1 plants continued to grow and successfully produced seeds, whereas most of the other plants (Wt and V) did not recover and died from drought, showing survival ratios of 18 % for Wt and 27 % for V (Fig. 5c). This
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Fig. 2 The expression patterns of SlNAC1. a The relative expression level of SlNAC1 was measured in leaves, stems, and roots of S. liaotungensis using qRT-PCR. b Measured in leaves under drought stress. c Measured in leaves under salt stress. d Measured in leaves under cold stress. e Measured in leaves under ABA treatment using qRT-PCR. Seven-week-old S. liaotungensis plants were treated with 10 % PEG 6000 (drought), 200 mM NaCl (salt), and 100 lM ABA, and exposed to 4 °C (cold) for 0.5, 1, 3, 6, 12, and 24 h. Standard errors were calculated from three biological replicates in which
SlActin1 (an actin gene, accession number JX860282.1) transcripts were used as internal controls. The 2-DDCT method was used to measure the relative expression levels of the target gene in stressed and non-stressed leaves. Error bars represent standard error. *, **, Significantly different from expression level of control (0 h): *, P \ 0.05; **, P \ 0.01; lowercase indicates significant differences between various tissues based on a one-way analysis of variance test (P \ 0.05; n = 3)
Fig. 3 Subcellular localization assay of SlNAC1 in onion epidermal cells. Onion epidermal cells were bombarded with constructs carrying 35S::GFP (Vector) or 35S::GFP-SlNAC1 by a GJ-1000 Gene gun. 35S::GFP and 35S::GFP-SlNAC1 fusion proteins were transiently
expressed in onion epidermal cells and observed using a Nikon Ti-E laser-scanning confocal microscope. Images are dark fields for green fluorescence (a, d), bright field (b, e), and merged (c, f). Bar 50 lm
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Fig. 4 Transactivation activity analysis of SlNAC1 in yeast. A fusion protein of the GAL4 DNA-binding domain and full-length SlNAC1 was checked for its transactivation activity in yeast strain AH109. The
pGBKT7 vector was used as a negative control. Experiments were repeated three times independently, and the results were consistent
Fig. 5 SlNAC1 expression in transgenic plants and drought tolerance assays of SlNAC1 overexpression plants. a SlNAC1 expression in transgenic plants. b Photographs taken when the drought stress phenotypes appeared and 3 days after re-watering. c Percentage of surviving plants in drought tolerance assays; bars indicate standard errors. d Leaf water loss assay. Leaf weights were measured at the
indicated time points (n = 9). Curves were drawn based on the data from three independent experiments. Wild-type (Wt) and pEGAD (V) lines were used as control plants. N1 and N2 represent pEGADSlNAC1-1 and pEGAD-SlNAC1-2 plants, respectively. Error bars represent standard error. *, **, Significantly different from Wt: *, P \ 0.05; **, P \ 0.01
experiment was repeated three times with similar results. The results showed that high-level expression of SlNAC1 could enhance plant drought tolerance. Regulation of transpiration plays a vital role in plant response to drought stress. To address whether the SlNAC1-related enhanced drought tolerance was associated with transpiration, we compared the water loss ratio of detached leaves from different plants (N1, N2, Wt and V) that were subjected to air-drying treatment at 25 °C with 55 % humidity. We found that the leaf weight of N1 and N2 plants decreased at a slow rate comparable to the water
loss rates (Fig. 5d). This quantitative result was repeated three times. Overexpression of SlNAC1 enhanced high salinity tolerance of transgenic Arabidopsis SlNAC1 is induced by salt (Fig. 2c); therefore, we asked whether this induction correlated with the plant’s response to salinity. As shown in Fig. 6a, the germination rates (radical emergence) of N1 and N2 seeds were slightly higher than Wt and V plants on MS medium with 200 mM
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Fig. 6 High salinity assays of SlNAC1 overexpression plants. (a) Germination rates analysis. Seeds of wild-type (Wt), pEGAD (V), pEGAD-SlNAC1-1 (N1), and pEGAD-SlNAC1-2 (N2) lines were placed on MS medium containing 200 mM NaCl and germinated for 3 days. Percentages are means (n = 40–60 each) of three repeats ±SE. b Phenotypic comparison of root lengths. Wt, V, and N1 and N2 seeds were germinated and grown on MS medium with or
without 200 mM NaCl for 7 days. c Statistical comparison of root lengths. d Photographs of the salt-stressed phenotypes. e Percentage of survival plants in salt tolerance assays; bars indicate standard errors. Wt and V lines were used as control plants. Error bars represent standard error. *, **, Significantly different from Wt: *, P \ 0.05; **, P \ 0.01
NaCl. The germination rates of all untreated plants were the same (data not shown). To further correlate SlNAC1 function with plant sensitivity to NaCl during the postgermination stage, root elongation was analyzed for these plants. Seedlings (4 days old) were transferred to MS medium with or without 200 mM NaCl. After 7 days, growth retardation was observed for N1 and N2 on MS medium; however, the retardation was not dramatic compared with Wt and V plants on MS medium with 200 mM NaCl (Fig. 6d, e). To characterize the function of SlNAC1 in salt tolerance, a whole-plant salt tolerance assay was performed in soil. Seedlings (10 days old) were transferred from MS plates to water-saturated soil for 7 days, and were then watered with 200 mM NaCl solution until severe salt-stressed phenotypes appeared (Fig. 6b). Most of the Wt and V plants died, showing low survival ratios (17 % for Wt, 17 % for V), but 39 % of the N1 and 35 % of the N2 lines continued to grow (Fig. 6c). The experiment was repeated three times with similar results. These
results indicated that high levels of expression of SlNAC1 could enhance plant salt tolerance.
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Overexpression of SlNAC1 enhanced cold tolerance of transgenic Arabidopsis SlNAC1 is induced by cold (Fig. 2d); therefore, we asked whether this induction correlated with the plant’s response to cold. To characterize the function of SlNAC1 in cold tolerance, a whole-plant cold tolerance assay was performed in soil. Seedlings (10 days old) were transferred from MS plates to water-saturated soil for 20 days and into -20 °C refrigerator for 90 min until severe cold-stressed phenotypes appeared (Fig. 7a). Most of the Wt and V plants died, showing low survival ratios (31 % for Wt, 35 % for V); but 75 % of the N1 and 57 % of the N2 lines continued to grow (Fig. 7b). The experiment was repeated three times with similar results. These results indicated that high levels of expression of SlNAC1 could enhance plant cold tolerance.
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Fig. 7 Cold tolerance assays of SlNAC1 overexpression plants. a Photographs taken after 3 days from the cold stress phenotypes that appeared. b Percentage of surviving plants in cold tolerance assays; bars indicate standard errors. Wild-type (Wt) and pEGAD
(V) lines were used as control plants. N1 and N2 represent pEGADSlNAC1-1 and pEGAD-SlNAC1-2 plants, respectively. Error bars represent standard error. *, **, Significantly different from Wt: *, P \ 0.05; **, P \ 0.01
Fig. 8 Sensitivity of SlNAC1 overexpression lines to ABA. a Germination rates of SlNAC1 overexpression lines on MS medium with 10 lM ABA. b, c Phenotypic and statistical comparisons of root lengths of wild-type, pEGAD (V), pEGAD-SlNAC1-1 (N1), and
pEGAD-SlNAC1-2 (N2) lines in the presence of MS with or without 10 lM ABA. Error bars represent standard error. *, **, Significantly different from Wt: *, P \ 0.05; **, P \ 0.01
SlNAC1 overexpression affects transgenic Arabidopsis sensitivity to ABA
was lower for N1 and N2 than Wt and V plants (Fig. 8b, c). Taken together, these results indicate that SlNAC1 modulates plant ABA signaling differently in the germination stage and root elongation stage; high SlNAC1 expression contributes to ABA hypersensitivity in transgenic Arabidopsis during germination, but the sensitivity was lower to Wt and V plants during the root elongation stage.
The expression of SlNAC1 was dramatically induced by ABA, as assessed by RT-qPCR (Fig. 2e), suggesting the potential function of SlNAC1 in the ABA response. To elucidate the role of SlNAC1 in ABA signaling, N1, N2, Wt, and V plants were germinated on MS plates with 10 lM ABA for 3 days. The high concentration of ABA (10 lM) used in this study led to extreme growth inhibition for all the plants; however, N1 and N2 plants showed the most severe arrested growth under these conditions, as reflected by lower germination rates. By contrast, the germination rates of Wt and V plants were higher (Fig. 8a). To further correlate SlNAC1 function with plant sensitivity to ABA during the post-germination stage, root elongation inhibition was analyzed for these plants. Seedlings (4 days old) were transferred to MS medium with or without 10 lM ABA. After 7 days, root growth inhibition by ABA
Discussion SlNAC1 may be an NAC transcription factor TFs are proteins that act together with other transcriptional regulators, including chromatin remodeling/modifying proteins, to encourage or obstruct the access of RNA polymerases to the DNA template (Udvardi et al. 2007). NAC proteins share a well-conserved N-terminal NAC domain (*150 amino acids; aa) and a diversified C-terminal
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transcription regulatory (TR) region. The NAC domain can be divided into five subdomains (A–E). The transcription regulatory regions can either activate or repress transcription (Puranik et al. 2012). In this study, we found that the SlNAC1 gene encodes a protein with a conserved NAC domain at its N terminus (supplemental data 1). Sequence alignment also revealed the characteristic conserved amino acid sequences (Fig. 1a). Phylogenetic analysis revealed that SlNAC1 was clustered into the TIP subgroup of Subfamily I (Fig. 1b). Subcellular localization experiments in onion epidermal cells indicated that SlNAC1 was localized in the nucleus (Fig. 3) and transactivation assays demonstrated that SlNAC1 functioned as a transcriptional activator (Fig. 4). These results may support the hypothesis that SlNAC1 is a novel member of NAC transcription factor family. SlNAC1 functions in ABA signaling and abiotic stress responses ABA is a broad-spectrum phytohormone involved not only in regulating growth and development, but also in coordinating various stress signal transduction pathways in plants during abiotic stresses. The plant response to abiotic stresses involves both ABA-dependent and ABA-independent signaling pathways. The ABA-dependent and ABA-independent signal transduction pathways from stress signal perception to gene expression involve different transcription factors, such as DREB, MYC/MYB, AREB/ABF, NAC, and zinc finger (Agarwal and Jha 2010; Li et al. 2010). A maize stress-responsive NAC transcription factor, ZmSNAC1, confers enhanced tolerance to dehydration in transgenic Arabidopsis (Lu et al. 2012). Soybean NAC transcription factors, GmNAC11 and GmNAC20, promote abiotic stress tolerance and lateral root formation in transgenic plants. Overexpression of GmNAC20 enhances salt and freezing tolerance in transgenic Arabidopsis plants; however, GmNAC11 overexpression only improves salt tolerance (Hao et al. 2011). However, the function of SlNAC1 under abiotic stress was unknown. Here, we found that the expression of SlNAC1 was induced by drought, high salt, cold, and ABA (Fig. 2b–e). To further identify the function of SlNAC1 in response to abiotic stresses, we overexpressed SlNAC1 in transgenic Arabidopsis thaliana. The transgenic plants showed improved drought, high salt and cold tolerance, as assessed by a wholeplant abiotic stress assay (Figs. 5b, 6b, 7a). Analysis of the transpiration rate of detached leaves indicated that the rate of water loss of the transgenic plants was lower than that of Wt and V plants (Fig. 5d). Meanwhile, the survival rate of transgenic plants was higher than that of Wt and V plants (Figs. 5c, 6c, 7b). These data provided evidence that SlNAC1 can enhance transgenic plants drought tolerance. In
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addition, we analyzed the germination rate and root elongation of SlNAC1 transgenic plants, Wt, and V plants under NaCl treatment. The germination ratio assay showed that the seeds of the transgenic plants had slightly higher germination rates compared with Wt and V plants under high salt stress (Fig. 6a). Moreover, root elongation assays indicated that overexpression of SlNAC1 led to root growth and no retardation in transgenic plants under normal conditions, and the retardation was not dramatic compared with Wt and V plants under high salt treatment. On the contrary, the root lengths of transgenic plants were longer (Fig. 6d, e). This evidence suggested that SlNAC1 transgenic plants had high salt tolerance. To further elucidate the role of SlNAC1 in ABA signaling, the germination ratio and root elongation were also tested under ABA treatment. The results showed that overexpression of SlNAC1 resulted in a significantly lower germination rate of transgenic plants seeds compared with Wt and V plants under ABA treatment, which indicated that high SlNAC1 expression contributed to ABA hypersensitivity in Arabidopsis during germination. However, this sensitivity was noticeably high during the root elongation stage; the root retardation of transgenic plants was not as dramatic as that observed for Wt and V plants (Fig. 8). This suggested that SlNAC1 might function in the ABA-dependent signaling pathway. Taken together, the results of the present study revealed that transcription of SlNAC1 was induced by various abiotic stresses, such as drought, high salt, cold, and ABA, and overexpression of SlNAC1 enhanced the tolerance of transgenic plants to drought, high salt, and cold. SlNAC1 may function in the ABA-dependent signaling pathway. SlNAC1 is an important gene and has significant research value As shown in Fig. 1b, SlNAC1 was clustered into the TIP subgroup of Subfamily I. There is some information about function of stress tolerance in TIP subgroup. Cold activation of a plasma membrane-tethered NAC transcription factor AtNAC62 (NTL6/ANAC062) induces a pathogen resistance response in Arabidopsis (Seo et al. 2010). Moreover, SnRK2.8 (sucrose non-fermenting related kinase 2.8)-mediated protein phosphorylation, in addition to a proteolytic processing event, is important for NTL6 function in inducing a drought-resistance response (Kim et al. 2012). Recent studies have also shown that ZmSNAC1 binds to the ABA-box of ZmOST1 [OPEN STOMATA 1 (OST1) ortholog in Zea mays], which is conserved in SnRK2s activated by ABA and is part of the contact site for the negative-regulating clade A PP2C phosphatases (Vilela et al. 2013). Another member of TIP subgroup, AtTIP, is an essential component in the TCV resistance response pathway (Ren et al. 2000). This
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suggests that further research is required to determine whether SlNAC1 is responsible for its response to biotic stresses, and whether the ABA-dependent signaling pathway of SlNAC1 is associated to SnRK2s. Thus, functional research of SlNAC1 has important biological significance and may provide more clues to stress tolerance research in plants. Acknowledgments We would like to thank Dr Shaoming Tong (Liaoning Normal University) for kindly providing the pEGAD vector and seeds of Arabidopsis thaliana and Dr Qiao Su (Dalian University of Technology) for providing the GeneGun (GJ-1000, China). This work was supported by grants from the National Natural Science Foundation of China (No. 31340052) and Liaoning Provincial Natural Science Foundation of China (2013020069).
References Agarwal P, Jha B (2010) Transcription factors in plants and ABA dependent and independent abiotic stress signalling. Biol Plant 54:201–212 Agarwal PK, Agarwal P, Reddy MK, Sopory SK (2006) Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep 25:1263–1274 Aida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M (1997) Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell 9:841–857 Chen X, Wang Y, Lv B, Li J, Luo L, Lu S, Zhang X, Ma H, Ming F (2014) The NAC family transcription factor OsNAP confers abiotic stress response through the ABA pathway. Plant Cell Physiol Chinnusamy V, Schumaker K, Zhu JK (2004) Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. J Exp Bot 55:225–236 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743 Dracup M, Turner N, Tang C, Reader M, Palta J, Gladstones J, Atkins C, Hamblin J (1998) Responses to abiotic stresses. Lupins as crop plants: biology, production and utilization. Wallingford, UK, 1998, pp 227–262 Fujita M, Fujita Y, Maruyama K, Seki M, Hiratsu K, Ohme-Takagi M, Tran L-SP, Yamaguchi-Shinozaki K, Shinozaki K (2004) A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway. Plant J 39:863–876 Hao YJ, Wei W, Song QX, Chen HW, Zhang YQ, Wang F, Zou HF, Lei G, Tian AG, Zhang WK, Ma B, Zhang JS, Chen SY (2011) Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants. Plant J 68:302–313 Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong L (2006) Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. PNAS USA 103:12987–12992 Jensen MK, Kjaersgaard T, Nielsen MM, Galberg P, Petersen K, O’Shea C, Skriver K (2009) The Arabidopsis thaliana NAC transcription factor family: structure–function relationships and determinants of ANAC019 stress signalling. Biochem J 426:183–196 Kim MJ, Park MJ, Seo PJ, Song JS, Kim HJ, Park CM (2012) Controlled nuclear import of the transcription factor NTL6
777 reveals a cytoplasmic role of SnRK2.8 in the drought-stress response. Biochem J 448:353–363 Li C, Lv J, Zhao X, Ai X, Zhu X, Wang M, Zhao S, Xia G (2010) TaCHP: a wheat zinc finger protein gene down-regulated by abscisic acid and salinity stress plays a positive role in stress tolerance. Plant Physiol 154:211–221 Liu G, Li X, Jin S, Liu X, Zhu L, Nie Y, Zhang X (2014) Overexpression of Rice NAC Gene SNAC1 improves drought and salt tolerance by enhancing root development and reducing transpiration rate in transgenic cotton. PLoS One 9:e86895 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta CT) Method. Methods 25:402–408 Lu M, Ying S, Zhang DF, Shi YS, Song YC, Wang TY, Li Y (2012) A maize stress-responsive NAC transcription factor, ZmSNAC1, confers enhanced tolerance to dehydration in transgenic Arabidopsis. Plant Cell Rep 31:1701–1711 Mao X, Chen S, Li A, Zhai C, Jing R (2014) Novel NAC transcription factor TaNAC67 confers enhanced multi-abiotic stress tolerances in Arabidopsis. PLoS ONE 9:e84359 Mauch-Mani B, Flors V (2009) The ATAF1 transcription factor: at the convergence point of ABA-dependent plant defense against biotic and abiotic stresses. Cell Res 19:1322–1323 Mukhopadhyay A, Vij S, Tyagi AK (2004) Overexpression of a zincfinger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco. Proc Natl Acad Sci USA 101:6309–6314 Nakashima K, Takasaki H, Mizoi J, Shinozaki K, YamaguchiShinozaki K (2011) NAC transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819:97–103 Puranik S, Sahu PP, Srivastava PS, Prasad M (2012) NAC proteins: regulation and role in stress tolerance. Trends Plant Sci 17:369–381 Ren T, Qu F, Morris TJ (2000) HRT gene function requires interaction between a NAC protein and viral capsid protein to confer resistance to turnip crinkle virus. Plant Cell 12:1917–1926 Seki M, Kamei A, Yamaguchi-Shinozaki K, Shinozaki K (2003) Molecular responses to drought, salinity and frost: common and different paths for plant protection. Curr Opin Biotechnol 14:194–199 Seo PJ, Kim MJ, Park JY, Kim SY, Jeon J, Lee YH, Kim J, Park CM (2010) Cold activation of a plasma membrane-tethered NAC transcription factor induces a pathogen resistance response in Arabidopsis. Plant J 61:661–671 Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410–417 Souer E, van Houwelingen A, Kloos D, Mol J, Koes R (1996) The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell 85:159–170 Takasaki H, Maruyama K, Kidokoro S, Ito Y, Fujita Y, Shinozaki K, Yamaguchi-Shinozaki K, Nakashima K (2010) The abiotic stress-responsive NAC-type transcription factor OsNAC5 regulates stress-inducible genes and stress tolerance in rice. Mol Genet Genomics 284:173–183 Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50:571–599 Tran LS, Nakashima K, Sakuma Y, Simpson SD, Fujita Y, Maruyama K, Fujita M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Isolation and functional analysis of Arabidopsis stressinducible NAC transcription factors that bind to a droughtresponsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell 16:2481–2498
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778 Udvardi MK, Kakar K, Wandrey M, Montanari O, Murray J, Andriankaja A, Zhang JY, Benedito V, Hofer JM, Chueng F, Town CD (2007) Legume transcription factors: global regulators of plant development and response to the environment. Plant Physiol 144:538–549 Vilela B, Moreno-Cortes A, Rabissi A, Leung J, Pages M, Lumbreras V (2013) The maize OST1 kinase homolog phosphorylates and regulates the maize SNAC1-type transcription factor. PLoS One 8:e58105 Wu Y, Deng Z, Lai J, Zhang Y, Yang C, Yin B, Zhao Q, Zhang L, Li Y, Xie Q (2009) Dual function of Arabidopsis ATAF1 in abiotic and biotic stress responses. Cell Res 19:1279–1290
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Plant Cell Rep (2014) 33:767–778 Yanhui C, Xiaoyuan Y, Kun H, Meihua L, Jigang L, Zhaofeng G, Zhiqiang L, Yunfei Z, Xiaoxiao W, Xiaoming Q (2006) The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol Biol 60:107–124 Zheng X, Chen B, Lu G, Han B (2009) Overexpression of a NAC transcription factor enhances rice drought and salt tolerance. Biochem Biophys Res Commun 379:985–989 Zhu J-K (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247
ORIGINAL PAPER
A novel NAC transcription factor from Suaeda liaotungensis K. enhanced transgenic Arabidopsis drought, salt, and cold stress tolerance Xiao-lan Li • Xing Yang • Yu-xin Hu Xiao-dong Yu • Qiu-li Li
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Received: 3 January 2014 / Revised: 28 February 2014 / Accepted: 12 March 2014 / Published online: 29 March 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Key message SlNAC1 functions as a stress-responsive NAC protein involved in the abscisic acid-dependent signaling pathway and enhances transgenic Arabidopsis drought, salt, and cold stress tolerance. Abstract NAC (NAM, ATAF1, 2, CUC2) transcription factors constitute the largest families of plant-specific transcription factors, known to be involved in various growth or developmental processes and in regulation of response to environmental stresses. However, only little information regarding stress-related NAC genes is available in Suaeda liaotungensis K. In this study, we cloned a full-length NAC gene (1,011 bp) named SlNAC1 using polymerase chain reaction from Suaeda liaotungensis K. and investigated its function by overexpression in transgenic Arabidopsis. SlNAC1 contains an NAC-conserved Communicated by Q. Zhao.
Electronic supplementary material The online version of this article (doi:10.1007/s00299-014-1602-y) contains supplementary material, which is available to authorized users. X. Li X. Yang Y. Hu X. Yu Q. Li (&) College of Life Sciences, Liaoning Normal University; Key Laboratory of Plant Biotechnology in Liaoning Province, 1 South Liushu Street, Ganjingzi District, Dalian 116081, Liaoning, China e-mail: [email protected] X. Li e-mail: [email protected] X. Yang e-mail: [email protected] Y. Hu e-mail: [email protected] X. Yu e-mail: [email protected]
domain. Its expression in S. liaotungensis was induced by drought, high-salt, and cold (4 °C) stresses and by abscisic acid. Subcellular localization experiments in onion epidermal cells indicated that SlNAC1 is localized in the nucleus. Yeast one-hybrid assays showed that SlNAC1 functions as a transcriptional activator. SlNAC1 transgenic Arabidopsis displayed a higher survival ratio and lower rate of water loss under drought stress; a higher germination ratio, higher survival ratio, and lower root inhibition rate under salt stress; a higher survival ratio under cold stress; and a lower germination ratio and root inhibition rate under abscisic acid treatment, compared with wildtype Arabidopsis. These results suggested that SlNAC1 functions as a stress-responsive NAC protein involved in the abscisic acid-dependent signaling pathway and may have potential applications in transgenic breeding to enhance crops’ abiotic stress tolerances. Keywords Abiotic stress NAC transcription factor Transgenic plant Stress resistance Suaeda liaotungensis K. Abbreviations ABA Abscisic acid CBF Core binding factor DREB Dehydration response element binding factor GFP Green fluorescent protein MYB Myeloblastosis NAC NAM (no apical meristem), ATAF1 or ATAF2 and CUC2 (cup-shaped cotyledon) ORF Open reading frame PEG Polyethylene glycol 6000 6000 RT-qPCR Real-time quantitative polymerase chain reaction
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SNAC TFs
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Stress-responsive NAC Transcription factors
Introduction Environment stresses, such as drought, high salinity, and cold, reduce productivity and cause significant crop losses globally. Drought and salinity affect more than 10 % of arable land, which results in more than 50 % decline in the average yields of important crops worldwide (Dracup et al. 1998). Tolerance or susceptibility to these stresses is a complex process, as stress may affect multiple stages of plant development and often several stresses concurrently affect the plants (Chinnusamy et al. 2004). Therefore, plants have evolved a series of mechanisms to survive such environmental adversities. Plant stress tolerance involves changes at whole-plant, tissue, cellular, physiological, and molecular levels. Molecular responses to abiotic stress include perception, signal transduction, gene expression and, ultimately, metabolic changes in the plant, resulting in stress tolerance (Agarwal et al. 2006). Transcription factors (TFs) and their corresponding cis-regulatory sequences act as molecular switches for gene expression, regulating their temporal and spatial expression. Increasing evidence demonstrates that numerous transcription factors, such as DREB, CBF, bZIP, zinc finger, MYB, and NAC, are directly or indirectly involved in the regulation of plant defense and stress responses (Thomashow 1999; Zhu 2002; Seki et al. 2003; Shinozaki et al. 2003; Fujita et al. 2004; Mukhopadhyay et al. 2004; Yanhui et al. 2006). NAC (NAM, ATAF1, 2, CUC2) transcription factors constitute the largest families of plant-specific transcription factors and the family is present in a wide range of land plants. NAC was derived from the names of the first three described TFs containing NAC domain, namely NAM (no apical meristem), ATAF1-2, and CUC2 (cup-shaped cotyledon) (Souer et al. 1996; Aida et al. 1997). The NAC TFs are associated with diverse processes, which include various developmental programs, senescence, formation of secondary walls, and biotic and abiotic stress response (Puranik et al. 2012). Stress-inducible NAC genes have been shown to be involved in abiotic stress tolerance (Nakashima et al. 2011). Transgenic plant overexpressing stress-responsive NAC (SNAC) genes have exhibited at least one type of improved abiotic stress tolerance (Nakashima et al. 2011). Arabidopsis ATAF1, ANAC019, ANAC055, and ANAC072 were found to be induced by drought, high salinity, and abscisic acid (ABA), and their
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overexpression in transgenic plants showed remarkably enhanced plant tolerance to drought (Wu et al. 2009; Mauch-Mani and Flors 2009; Jensen et al. 2009; Tran et al. 2004). Rice ONAC045, OsNAC5, OsNAP, SNAC1, wheat TaNAC67, and maize ZmSNAC1 were reported to be drought, high-salt, and low-temperature stresses, and abscisic acid (ABA)-inducible genes. Plants individually overexpressing these genes showed an increase in drought tolerance or salt tolerance, or low temperature tolerance (Zheng et al. 2009; Takasaki et al. 2010; Chen et al. 2014; Hu et al. 2006; Liu et al. 2014; Mao et al. 2014; Lu et al. 2012). These studies indicate that SNAC transcription factors have important roles for the control of abiotic stress tolerance and that their overexpression can improve transgenic plants stress tolerance. However, there is no information on the abiotic stressrelated NAC proteins in Suaeda liaotungensis K, a halophytic plant, which grows widely by the seashore in Dalian, China, as a source of genetic material for introduction into non-salt-tolerant crops. We discovered a sequence annotated as an NAC transcription factor from the database of S. liaotungensis transcriptome sequencing and named this protein SlNAC1. To identify whether SlNAC1 was related to abiotic stress, we designed preliminary tests and found that SlNAC1 was induced by salt stress. In this study, we cloned SlNAC1 and characterized its function. A search for the conserved domains in NCBI showed that SlNAC1 contained an NAC domain. The expression of SlNAC1 was induced by drought, high salt, cold, and abscisic acid (ABA), as assessed by real-time quantitative polymerase chain reaction. Its overexpression improved the drought, high-salt, and cold stress tolerance of transgenic plants. Furthermore, SlNAC1 is localized in the nucleus and functions as a transcriptional activator, as determined by subcellular localization and transactivation assays. Therefore, SlNAC1 may become a hot topic for abiotic stress resistance research and is expected to be used in genetically modified crops.
Materials and methods Plant materials Green leaves and seeds of wild S. liaotungensis were collected from the seashore in Dalian. The leaves were used for SlNAC1 cDNA cloning. The seeds were used to breed seedlings for gene expression analysis. Arabidopsis thaliana accession Columbia (Col-0) was used for gene transformation and as a phenotypic control and was grown in a controlled environment chamber at 22 °C, with a 16/8 photoperiod, light intensity of 120–150 lmol m-2 s-1, and 60 % relative humidity.
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SlNAC1 isolation and sequence analysis The sequence of SlNAC1 (GenBank accession number KC176074) was obtained from the database of S. liaotungensis transcriptome sequencing. It was 1,011-bp long and contained a full-length open reading frame (ORF). To obtain the ORF by the reverse transcription polymerase chain reaction (RT-PCR) approach, two primers were used (sense: 50 -CTCTCTATCTCACCTAAAGTTTG C-30 and antisense: 50 -GGAAGTCAATCATGGAATCTCTAC-30 ). The PCR conditions for amplifying SlNAC1 were as follows: 3 min pre-denaturation at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 50 °C, 1 min at 72 °C; and a final extension for 10 min at 72 °C. The PCR product was purified and cloned into the pEASY-T1 cloning vector (TRANSGEN BIOTECH, China) for sequencing (Takara, Japan). Alignment of SlNAC1 and other NAC proteins was performed using the ClustalX program (version 1.83) and viewed by GeneDoc software (version 2.5). A phylogenetic tree was constructed using the MEGA program (version 4.0) by the neighbor-joining method. The parameters of pairwise deletion and the p-distance model were used. A bootstrap test of phylogeny was performed with 1,000 replicates.
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was conducted on a Thermal Cycler Dice Real Time System TP800 (Takara, Japan) using SYBR Premix Ex Taq II (Tli RnaseH Plus) (Takara, Japan). Baseline and threshold cycles (Ct) were automatically determined using the Thermal Cycler Dice Real Time System TP800 Software release Version 3.0. Relative gene expression with respect to SlActin was determined as described previously, using the 2 (-DD CT) method (Livak and Schmittgen 2001). Subcellular localization of SlNAC1 The full-length ORF of SlNAC1 was amplified with the primers 50 -gcGAATTCATGGGAGCTTTAATTATAA GAGA A-30 and 50 -gcGGATCCCTACCAATCATGT CTTGTATT GC-30 , digested with EcoRI and BamHI, and fused to the 30 end of GFP in the pEGAD vector to generate the fusion construct 35S::GFP-SlNAC1. The fusion gene and the negative control pEGAD vector were transformed into living onion epidermal cells by biolistic bombardment with a GeneGun (GJ-1000; China), according to the instruction manual (helium pressure, 9 MPa). Protein expression was observed under a confocal laser-scanning microscope (Ti-E; Nikon). Transactivation assay of SlNAC1
Expression analysis of SlNAC1 under different treatments Seedlings (4 weeks old) of S. liaotungensis were used for the various stress treatments. The treatments were executed at 25 °C and 50–65 % humidity and the seedlings were grown in 1/10 MS culture medium with 200 mM NaCl, 100 lM ABA, and 10 % PEG 6000 (polyethylene glycol 6000), respectively. For cold treatment, seedlings were transferred to a growth chamber at a temperature of 4 °C. For gene expression analysis, total RNA was isolated from liquid nitrogen-frozen seedlings using RNAiso Plus reagent (Takara, Japan), according to the manufacturer’s protocol. RNA integrity was verified by 1 % agar gel electrophoresis. The first-strand cDNA was synthesized with 1 lg of total RNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan), according to the manufacturer’s procedure. Primers were designed using Beacon Designer version 7.0 (Premier Biosoft international, Palo Alto, CA, USA) with melting temperatures of 60–65 °C, primer lengths of 20–24 bp, and amplicon lengths of 90–200 bp. The primers of SlNAC1 and the internal reference gene SlActin (a S. liaotungensis actin gene, GenBank accession number: JX860282.1) used for qRT-PCR were 50 -AGCGTACTCACTGGGTTATCCAT30 and 50 -ATCGGCAAAGAACAAAAGCAC-30 , and 50 ATCCCAAGGCTAATCGTGAAAA-30 and 50 -CAC 0 CATCACCAGAGTCCAACA-3 , respectively. qRT-PCR
The entire coding region of SlNAC1 was fused to the GAL4 DNA-binding domain in the pGBKT7 vector. According to the manufacturer’s protocol (Stratagene, USA), pGBKT7SlNAC1 and the negative control pGBKT7 vector were transferred into the yeast strain AH109, respectively. The transformed yeast cells were grown on SD glucose medium lacking Trp, and medium lacking Trp, His, and Ade, respectively. The transactivation activity of each protein was evaluated according to its growth status and b-galactosidase filter lift assay (Yeast Protocols Handbook; Clontech, USA). Abiotic stress assays of transgenic Arabidopsis plants The transformation vectors harboring the 35S::GFP or 35S::GFP-SlNAC1 constructs were introduced into Agrobacterium tumefaciens (GV3101) and then transferred into wild-type (Wt) Arabidopsis by the floral dip transformation method (Clough and Bent 1998). Positive transgenic lines of T1 and T2 generations were screened on MS agar medium containing 37.5 lM Basta. The T2 generation was selected according to its segregation ratio (resistant:sensitive = 3:1), and confirmed by genomic PCR. The selected transgenic lines (pEGAD-SlNAC1-1 and pEGADSlNAC1-2, N1 and N2) that displayed the segregation ratio were chosen for further analysis. Expression levels of SlNAC1 were determined by semi-quantitative RT-PCR.
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Total RNA samples were extracted from appropriate plant materials using the RNAiso Plus kit (Takara, Japan). Synthesis of first cDNA, RT-PCR was performed as described above. Expression levels were quantitated by agar gel electrophoresis. T2 generation seeds were sterilized, suspended in 0.1 % agar, and plated on MS medium (including 37.5 lM Basta), then stratified in darkness at 4 °C for 2 days and transferred to growth chambers with the same environmental conditions as described above. Ten-day-old seedlings were transferred from MS plates to water-saturated soil for 7 days, water was withheld from one group until the plants showed evident drought-stressed phenotypes and then the plants were re-watered for drought stress treatment. A second group was watered with 200 mM NaCl solution until the plants showed evident salt-stressed phenotypes for high salt stress treatment. A third group was grown for another 2 weeks, was treated at -20 °C for 90 min, and transferred to normal conditions. For the leaf water loss assay, rosette leaves excised from plants grown in soil were placed on filter paper for air-drying treatment. The leaf weight was measured over a series of time points. To conduct the root growth assay, seeds sown on MS plates were stratified for 2 days at 4 °C and grown vertically for 3 days under normal conditions, before the seedlings were transferred to vertical square MS plates with ABA (10 lM) or NaCl (200 mM). To conduct the germination rate assay, seeds of transgenic lines (N1 and N2) and control lines (wild-type and vector control) were placed on MS medium without or with ABA (10 lM), NaCl (200 mM) and 37.5 lM Basta, respectively. Seeds were vernalized at 4 °C in the dark for 3 days and then transferred to growth chambers. The germination ratio was calculated on the sixth day. Statistical analysis For qRT-PCR, the 2 -DD CT method was used to calculate relative expression levels of the target gene in stressed and non-stressed leaves and other tissues. Significant differences in relative expression levels were identified using a one-way analysis of variance test (P = 0.05; n = 3) using SPSS 13.0 (SPSS Inc., USA).
Results
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presence of the NAC domain in SlNAC1 (Fig. 1a). Phylogenetic analysis revealed that SlNAC1 was clustered into the TIP subgroup of Subfamily I (Fig. 1b). The proteins used in the alignment and phylogenetic tree all had an NAC domain and were obtained by database searching in NCBI. SlNAC1 is a potential stress-related gene To identify whether SlNAC1 could be induced by abiotic stresses, the expression pattern of SlNAC1 in different tissues of wild S. liaotungensis and under various abiotic stress treatments in young leaves was investigated using RT-qPCR. The results showed that the expression of SlNAC1 was the highest in the leaves (Fig. 2a) and that SlNAC1 was induced by drought (10 % PEG 6000) (Fig. 2b), high salt (200 mM NaCl) (Fig. 2c), cold (4 °C) (Fig. 2d), and 100 lM ABA (Fig. 2e). The expression patterns and maximum expression levels differed for each stress treatment. The relative expression levels peaked at 0.5 h for drought, 0.5 h for NaCl, 3 h for cold, and 1 h for ABA, with the corresponding maxima being 2.82-, 9.28-, 6.28-, and 1.92-fold greater than the control (0 h), respectively. SlNAC1 functions as a transcriptional activator and is localized in the nucleus To determine the subcellular localization of SlNAC1, a GFP-SlNAC1 fusion construct and the GFP control in pEGAD driven by the CaMV 35S promoter were transiently expressed in onion epidermal cells and analyzed by confocal laser-scanning microscopy. Figure 3a–c shows that the GFP-SlNAC1 fusion protein was targeted to the nuclei of the cells, while the control GFP protein was located in the cytoplasm (Fig. 3d–f). This result indicated that SlNAC1 was a nuclear protein. The yeast one-hybrid system was used to identify a possible transcriptional activation function of SlNAC1. As shown in Fig. 4, all transformants grew well on SD/Trpmedium. However, only transformants containing pGBKT7-SlNAC1 could grow on SD/Trp-/His-/Ade-medium and showed b-galactosidase activity that cells containing pGBKT7 could not (Fig. 4). These results indicated that SlNAC1 functioned as a transcriptional activator. Overexpression of SlNAC1 confers drought tolerance on transgenic Arabidopsis
SlNAC1 encodes a protein with an NAC domain The SlNAC1 gene encodes a 336-amino acid protein with a conserved NAC domain containing five subdomains at its N terminus (supplemental data 1). Multiple alignments with NAC proteins from other species confirmed the
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We showed that SlNAC1 is a drought-inducible gene (Fig. 2b); therefore it is likely to regulate drought signaling. To characterize such a function, a whole-plant drought assay was performed in soil using T2 generation transgenic lines to test SlNAC1 expression levels (Fig. 5a). Seedlings
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Fig. 1 Multiple alignment of SlNAC1 and phylogenetic tree analysis. a Multiple alignments of NAC proteins from TIP subgroup. Identical amino acid residues are highlighted in red. The regions annotated indicate the subdomains of NAC domain. b Phylogenetic tree of NAC proteins from different species. SlNAC1 was labeled with a big red dot. All of the proteins used in the phylogenetic tree came from database of NCBI. The corresponding accession numbers of the names are as follows: CaNAC6, gi|242877185|; AtNAC36, gi|145360022|; NtTERN, gi|4996349|; AtNTM1, gi|145332955|; ANAC001, gi|297337931|; AtNAC089, gi|75165521|; AtNAC60, gi|332644364|; HvTPA, gi|371925001|; OsNAC8, gi|6730946|; ZmTPA, gi|414876898|; AtVND1, gi|15227958|; OsNAC7, gi|6730944|; AtNAC014, gi|42571727|; AtNAC62, gi|15229161|;
AtTIP, gi|30689531|; MtNAC969, gi|359360012|; AtNAP, gi|18409291|; AtNAC2, gi|6456751|; AtSENU5,gi|9955562|; AhNAC2, gi|209171097|; AhNAC3, gi|209171095|; AtRD26, gi|18416983|; AtNAC019, gi|15219112|; AtNAC055, gi|15232604|; ATAF1, gi|15223456|; GmNAC2, gi|66394512|; AmNAC1, gi|154362215|; SsNAC23, gi|58013003|; ZmNAC1, gi|165855636|; SNAC2, gi|311815437|; TaNAC3, gi|296044564|; SNAC1, gi|88770831|; OsNAC3, gi|6730936|; Ta296044566, gi|296044566|; TaNAC7, gi|292659254|; AtNAC1, gi|6649236|; Zm413953607, gi|413953607|; ANAC011, gi|297336836|; AtCUC1, gi|15232511|; AtNAM, gi|6456751|; TaNAC8, gi|292659256|; TaNAM, gi|292659395|; TaNTL5, gi|292659258| (color figure online)
(10 days old) were transferred from MS plates to watersaturated soil for 7 days, and then water was withheld to cause a severe water deficit in soil. After 21 days, most of the Wt, pEGAD [vector control (V)], and pEGAD-SlNAC1 (N1, N2) plants had wilted because of the extreme water
deprivation (Fig. 5b). After re-watering, 62–76 % of pEGAD-SlNAC1 plants continued to grow and successfully produced seeds, whereas most of the other plants (Wt and V) did not recover and died from drought, showing survival ratios of 18 % for Wt and 27 % for V (Fig. 5c). This
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Fig. 2 The expression patterns of SlNAC1. a The relative expression level of SlNAC1 was measured in leaves, stems, and roots of S. liaotungensis using qRT-PCR. b Measured in leaves under drought stress. c Measured in leaves under salt stress. d Measured in leaves under cold stress. e Measured in leaves under ABA treatment using qRT-PCR. Seven-week-old S. liaotungensis plants were treated with 10 % PEG 6000 (drought), 200 mM NaCl (salt), and 100 lM ABA, and exposed to 4 °C (cold) for 0.5, 1, 3, 6, 12, and 24 h. Standard errors were calculated from three biological replicates in which
SlActin1 (an actin gene, accession number JX860282.1) transcripts were used as internal controls. The 2-DDCT method was used to measure the relative expression levels of the target gene in stressed and non-stressed leaves. Error bars represent standard error. *, **, Significantly different from expression level of control (0 h): *, P \ 0.05; **, P \ 0.01; lowercase indicates significant differences between various tissues based on a one-way analysis of variance test (P \ 0.05; n = 3)
Fig. 3 Subcellular localization assay of SlNAC1 in onion epidermal cells. Onion epidermal cells were bombarded with constructs carrying 35S::GFP (Vector) or 35S::GFP-SlNAC1 by a GJ-1000 Gene gun. 35S::GFP and 35S::GFP-SlNAC1 fusion proteins were transiently
expressed in onion epidermal cells and observed using a Nikon Ti-E laser-scanning confocal microscope. Images are dark fields for green fluorescence (a, d), bright field (b, e), and merged (c, f). Bar 50 lm
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Fig. 4 Transactivation activity analysis of SlNAC1 in yeast. A fusion protein of the GAL4 DNA-binding domain and full-length SlNAC1 was checked for its transactivation activity in yeast strain AH109. The
pGBKT7 vector was used as a negative control. Experiments were repeated three times independently, and the results were consistent
Fig. 5 SlNAC1 expression in transgenic plants and drought tolerance assays of SlNAC1 overexpression plants. a SlNAC1 expression in transgenic plants. b Photographs taken when the drought stress phenotypes appeared and 3 days after re-watering. c Percentage of surviving plants in drought tolerance assays; bars indicate standard errors. d Leaf water loss assay. Leaf weights were measured at the
indicated time points (n = 9). Curves were drawn based on the data from three independent experiments. Wild-type (Wt) and pEGAD (V) lines were used as control plants. N1 and N2 represent pEGADSlNAC1-1 and pEGAD-SlNAC1-2 plants, respectively. Error bars represent standard error. *, **, Significantly different from Wt: *, P \ 0.05; **, P \ 0.01
experiment was repeated three times with similar results. The results showed that high-level expression of SlNAC1 could enhance plant drought tolerance. Regulation of transpiration plays a vital role in plant response to drought stress. To address whether the SlNAC1-related enhanced drought tolerance was associated with transpiration, we compared the water loss ratio of detached leaves from different plants (N1, N2, Wt and V) that were subjected to air-drying treatment at 25 °C with 55 % humidity. We found that the leaf weight of N1 and N2 plants decreased at a slow rate comparable to the water
loss rates (Fig. 5d). This quantitative result was repeated three times. Overexpression of SlNAC1 enhanced high salinity tolerance of transgenic Arabidopsis SlNAC1 is induced by salt (Fig. 2c); therefore, we asked whether this induction correlated with the plant’s response to salinity. As shown in Fig. 6a, the germination rates (radical emergence) of N1 and N2 seeds were slightly higher than Wt and V plants on MS medium with 200 mM
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Fig. 6 High salinity assays of SlNAC1 overexpression plants. (a) Germination rates analysis. Seeds of wild-type (Wt), pEGAD (V), pEGAD-SlNAC1-1 (N1), and pEGAD-SlNAC1-2 (N2) lines were placed on MS medium containing 200 mM NaCl and germinated for 3 days. Percentages are means (n = 40–60 each) of three repeats ±SE. b Phenotypic comparison of root lengths. Wt, V, and N1 and N2 seeds were germinated and grown on MS medium with or
without 200 mM NaCl for 7 days. c Statistical comparison of root lengths. d Photographs of the salt-stressed phenotypes. e Percentage of survival plants in salt tolerance assays; bars indicate standard errors. Wt and V lines were used as control plants. Error bars represent standard error. *, **, Significantly different from Wt: *, P \ 0.05; **, P \ 0.01
NaCl. The germination rates of all untreated plants were the same (data not shown). To further correlate SlNAC1 function with plant sensitivity to NaCl during the postgermination stage, root elongation was analyzed for these plants. Seedlings (4 days old) were transferred to MS medium with or without 200 mM NaCl. After 7 days, growth retardation was observed for N1 and N2 on MS medium; however, the retardation was not dramatic compared with Wt and V plants on MS medium with 200 mM NaCl (Fig. 6d, e). To characterize the function of SlNAC1 in salt tolerance, a whole-plant salt tolerance assay was performed in soil. Seedlings (10 days old) were transferred from MS plates to water-saturated soil for 7 days, and were then watered with 200 mM NaCl solution until severe salt-stressed phenotypes appeared (Fig. 6b). Most of the Wt and V plants died, showing low survival ratios (17 % for Wt, 17 % for V), but 39 % of the N1 and 35 % of the N2 lines continued to grow (Fig. 6c). The experiment was repeated three times with similar results. These
results indicated that high levels of expression of SlNAC1 could enhance plant salt tolerance.
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Overexpression of SlNAC1 enhanced cold tolerance of transgenic Arabidopsis SlNAC1 is induced by cold (Fig. 2d); therefore, we asked whether this induction correlated with the plant’s response to cold. To characterize the function of SlNAC1 in cold tolerance, a whole-plant cold tolerance assay was performed in soil. Seedlings (10 days old) were transferred from MS plates to water-saturated soil for 20 days and into -20 °C refrigerator for 90 min until severe cold-stressed phenotypes appeared (Fig. 7a). Most of the Wt and V plants died, showing low survival ratios (31 % for Wt, 35 % for V); but 75 % of the N1 and 57 % of the N2 lines continued to grow (Fig. 7b). The experiment was repeated three times with similar results. These results indicated that high levels of expression of SlNAC1 could enhance plant cold tolerance.
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Fig. 7 Cold tolerance assays of SlNAC1 overexpression plants. a Photographs taken after 3 days from the cold stress phenotypes that appeared. b Percentage of surviving plants in cold tolerance assays; bars indicate standard errors. Wild-type (Wt) and pEGAD
(V) lines were used as control plants. N1 and N2 represent pEGADSlNAC1-1 and pEGAD-SlNAC1-2 plants, respectively. Error bars represent standard error. *, **, Significantly different from Wt: *, P \ 0.05; **, P \ 0.01
Fig. 8 Sensitivity of SlNAC1 overexpression lines to ABA. a Germination rates of SlNAC1 overexpression lines on MS medium with 10 lM ABA. b, c Phenotypic and statistical comparisons of root lengths of wild-type, pEGAD (V), pEGAD-SlNAC1-1 (N1), and
pEGAD-SlNAC1-2 (N2) lines in the presence of MS with or without 10 lM ABA. Error bars represent standard error. *, **, Significantly different from Wt: *, P \ 0.05; **, P \ 0.01
SlNAC1 overexpression affects transgenic Arabidopsis sensitivity to ABA
was lower for N1 and N2 than Wt and V plants (Fig. 8b, c). Taken together, these results indicate that SlNAC1 modulates plant ABA signaling differently in the germination stage and root elongation stage; high SlNAC1 expression contributes to ABA hypersensitivity in transgenic Arabidopsis during germination, but the sensitivity was lower to Wt and V plants during the root elongation stage.
The expression of SlNAC1 was dramatically induced by ABA, as assessed by RT-qPCR (Fig. 2e), suggesting the potential function of SlNAC1 in the ABA response. To elucidate the role of SlNAC1 in ABA signaling, N1, N2, Wt, and V plants were germinated on MS plates with 10 lM ABA for 3 days. The high concentration of ABA (10 lM) used in this study led to extreme growth inhibition for all the plants; however, N1 and N2 plants showed the most severe arrested growth under these conditions, as reflected by lower germination rates. By contrast, the germination rates of Wt and V plants were higher (Fig. 8a). To further correlate SlNAC1 function with plant sensitivity to ABA during the post-germination stage, root elongation inhibition was analyzed for these plants. Seedlings (4 days old) were transferred to MS medium with or without 10 lM ABA. After 7 days, root growth inhibition by ABA
Discussion SlNAC1 may be an NAC transcription factor TFs are proteins that act together with other transcriptional regulators, including chromatin remodeling/modifying proteins, to encourage or obstruct the access of RNA polymerases to the DNA template (Udvardi et al. 2007). NAC proteins share a well-conserved N-terminal NAC domain (*150 amino acids; aa) and a diversified C-terminal
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transcription regulatory (TR) region. The NAC domain can be divided into five subdomains (A–E). The transcription regulatory regions can either activate or repress transcription (Puranik et al. 2012). In this study, we found that the SlNAC1 gene encodes a protein with a conserved NAC domain at its N terminus (supplemental data 1). Sequence alignment also revealed the characteristic conserved amino acid sequences (Fig. 1a). Phylogenetic analysis revealed that SlNAC1 was clustered into the TIP subgroup of Subfamily I (Fig. 1b). Subcellular localization experiments in onion epidermal cells indicated that SlNAC1 was localized in the nucleus (Fig. 3) and transactivation assays demonstrated that SlNAC1 functioned as a transcriptional activator (Fig. 4). These results may support the hypothesis that SlNAC1 is a novel member of NAC transcription factor family. SlNAC1 functions in ABA signaling and abiotic stress responses ABA is a broad-spectrum phytohormone involved not only in regulating growth and development, but also in coordinating various stress signal transduction pathways in plants during abiotic stresses. The plant response to abiotic stresses involves both ABA-dependent and ABA-independent signaling pathways. The ABA-dependent and ABA-independent signal transduction pathways from stress signal perception to gene expression involve different transcription factors, such as DREB, MYC/MYB, AREB/ABF, NAC, and zinc finger (Agarwal and Jha 2010; Li et al. 2010). A maize stress-responsive NAC transcription factor, ZmSNAC1, confers enhanced tolerance to dehydration in transgenic Arabidopsis (Lu et al. 2012). Soybean NAC transcription factors, GmNAC11 and GmNAC20, promote abiotic stress tolerance and lateral root formation in transgenic plants. Overexpression of GmNAC20 enhances salt and freezing tolerance in transgenic Arabidopsis plants; however, GmNAC11 overexpression only improves salt tolerance (Hao et al. 2011). However, the function of SlNAC1 under abiotic stress was unknown. Here, we found that the expression of SlNAC1 was induced by drought, high salt, cold, and ABA (Fig. 2b–e). To further identify the function of SlNAC1 in response to abiotic stresses, we overexpressed SlNAC1 in transgenic Arabidopsis thaliana. The transgenic plants showed improved drought, high salt and cold tolerance, as assessed by a wholeplant abiotic stress assay (Figs. 5b, 6b, 7a). Analysis of the transpiration rate of detached leaves indicated that the rate of water loss of the transgenic plants was lower than that of Wt and V plants (Fig. 5d). Meanwhile, the survival rate of transgenic plants was higher than that of Wt and V plants (Figs. 5c, 6c, 7b). These data provided evidence that SlNAC1 can enhance transgenic plants drought tolerance. In
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addition, we analyzed the germination rate and root elongation of SlNAC1 transgenic plants, Wt, and V plants under NaCl treatment. The germination ratio assay showed that the seeds of the transgenic plants had slightly higher germination rates compared with Wt and V plants under high salt stress (Fig. 6a). Moreover, root elongation assays indicated that overexpression of SlNAC1 led to root growth and no retardation in transgenic plants under normal conditions, and the retardation was not dramatic compared with Wt and V plants under high salt treatment. On the contrary, the root lengths of transgenic plants were longer (Fig. 6d, e). This evidence suggested that SlNAC1 transgenic plants had high salt tolerance. To further elucidate the role of SlNAC1 in ABA signaling, the germination ratio and root elongation were also tested under ABA treatment. The results showed that overexpression of SlNAC1 resulted in a significantly lower germination rate of transgenic plants seeds compared with Wt and V plants under ABA treatment, which indicated that high SlNAC1 expression contributed to ABA hypersensitivity in Arabidopsis during germination. However, this sensitivity was noticeably high during the root elongation stage; the root retardation of transgenic plants was not as dramatic as that observed for Wt and V plants (Fig. 8). This suggested that SlNAC1 might function in the ABA-dependent signaling pathway. Taken together, the results of the present study revealed that transcription of SlNAC1 was induced by various abiotic stresses, such as drought, high salt, cold, and ABA, and overexpression of SlNAC1 enhanced the tolerance of transgenic plants to drought, high salt, and cold. SlNAC1 may function in the ABA-dependent signaling pathway. SlNAC1 is an important gene and has significant research value As shown in Fig. 1b, SlNAC1 was clustered into the TIP subgroup of Subfamily I. There is some information about function of stress tolerance in TIP subgroup. Cold activation of a plasma membrane-tethered NAC transcription factor AtNAC62 (NTL6/ANAC062) induces a pathogen resistance response in Arabidopsis (Seo et al. 2010). Moreover, SnRK2.8 (sucrose non-fermenting related kinase 2.8)-mediated protein phosphorylation, in addition to a proteolytic processing event, is important for NTL6 function in inducing a drought-resistance response (Kim et al. 2012). Recent studies have also shown that ZmSNAC1 binds to the ABA-box of ZmOST1 [OPEN STOMATA 1 (OST1) ortholog in Zea mays], which is conserved in SnRK2s activated by ABA and is part of the contact site for the negative-regulating clade A PP2C phosphatases (Vilela et al. 2013). Another member of TIP subgroup, AtTIP, is an essential component in the TCV resistance response pathway (Ren et al. 2000). This
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suggests that further research is required to determine whether SlNAC1 is responsible for its response to biotic stresses, and whether the ABA-dependent signaling pathway of SlNAC1 is associated to SnRK2s. Thus, functional research of SlNAC1 has important biological significance and may provide more clues to stress tolerance research in plants. Acknowledgments We would like to thank Dr Shaoming Tong (Liaoning Normal University) for kindly providing the pEGAD vector and seeds of Arabidopsis thaliana and Dr Qiao Su (Dalian University of Technology) for providing the GeneGun (GJ-1000, China). This work was supported by grants from the National Natural Science Foundation of China (No. 31340052) and Liaoning Provincial Natural Science Foundation of China (2013020069).
References Agarwal P, Jha B (2010) Transcription factors in plants and ABA dependent and independent abiotic stress signalling. Biol Plant 54:201–212 Agarwal PK, Agarwal P, Reddy MK, Sopory SK (2006) Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep 25:1263–1274 Aida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M (1997) Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell 9:841–857 Chen X, Wang Y, Lv B, Li J, Luo L, Lu S, Zhang X, Ma H, Ming F (2014) The NAC family transcription factor OsNAP confers abiotic stress response through the ABA pathway. Plant Cell Physiol Chinnusamy V, Schumaker K, Zhu JK (2004) Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. J Exp Bot 55:225–236 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743 Dracup M, Turner N, Tang C, Reader M, Palta J, Gladstones J, Atkins C, Hamblin J (1998) Responses to abiotic stresses. Lupins as crop plants: biology, production and utilization. Wallingford, UK, 1998, pp 227–262 Fujita M, Fujita Y, Maruyama K, Seki M, Hiratsu K, Ohme-Takagi M, Tran L-SP, Yamaguchi-Shinozaki K, Shinozaki K (2004) A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway. Plant J 39:863–876 Hao YJ, Wei W, Song QX, Chen HW, Zhang YQ, Wang F, Zou HF, Lei G, Tian AG, Zhang WK, Ma B, Zhang JS, Chen SY (2011) Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants. Plant J 68:302–313 Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong L (2006) Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. PNAS USA 103:12987–12992 Jensen MK, Kjaersgaard T, Nielsen MM, Galberg P, Petersen K, O’Shea C, Skriver K (2009) The Arabidopsis thaliana NAC transcription factor family: structure–function relationships and determinants of ANAC019 stress signalling. Biochem J 426:183–196 Kim MJ, Park MJ, Seo PJ, Song JS, Kim HJ, Park CM (2012) Controlled nuclear import of the transcription factor NTL6
777 reveals a cytoplasmic role of SnRK2.8 in the drought-stress response. Biochem J 448:353–363 Li C, Lv J, Zhao X, Ai X, Zhu X, Wang M, Zhao S, Xia G (2010) TaCHP: a wheat zinc finger protein gene down-regulated by abscisic acid and salinity stress plays a positive role in stress tolerance. Plant Physiol 154:211–221 Liu G, Li X, Jin S, Liu X, Zhu L, Nie Y, Zhang X (2014) Overexpression of Rice NAC Gene SNAC1 improves drought and salt tolerance by enhancing root development and reducing transpiration rate in transgenic cotton. PLoS One 9:e86895 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta CT) Method. Methods 25:402–408 Lu M, Ying S, Zhang DF, Shi YS, Song YC, Wang TY, Li Y (2012) A maize stress-responsive NAC transcription factor, ZmSNAC1, confers enhanced tolerance to dehydration in transgenic Arabidopsis. Plant Cell Rep 31:1701–1711 Mao X, Chen S, Li A, Zhai C, Jing R (2014) Novel NAC transcription factor TaNAC67 confers enhanced multi-abiotic stress tolerances in Arabidopsis. PLoS ONE 9:e84359 Mauch-Mani B, Flors V (2009) The ATAF1 transcription factor: at the convergence point of ABA-dependent plant defense against biotic and abiotic stresses. Cell Res 19:1322–1323 Mukhopadhyay A, Vij S, Tyagi AK (2004) Overexpression of a zincfinger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco. Proc Natl Acad Sci USA 101:6309–6314 Nakashima K, Takasaki H, Mizoi J, Shinozaki K, YamaguchiShinozaki K (2011) NAC transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819:97–103 Puranik S, Sahu PP, Srivastava PS, Prasad M (2012) NAC proteins: regulation and role in stress tolerance. Trends Plant Sci 17:369–381 Ren T, Qu F, Morris TJ (2000) HRT gene function requires interaction between a NAC protein and viral capsid protein to confer resistance to turnip crinkle virus. Plant Cell 12:1917–1926 Seki M, Kamei A, Yamaguchi-Shinozaki K, Shinozaki K (2003) Molecular responses to drought, salinity and frost: common and different paths for plant protection. Curr Opin Biotechnol 14:194–199 Seo PJ, Kim MJ, Park JY, Kim SY, Jeon J, Lee YH, Kim J, Park CM (2010) Cold activation of a plasma membrane-tethered NAC transcription factor induces a pathogen resistance response in Arabidopsis. Plant J 61:661–671 Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410–417 Souer E, van Houwelingen A, Kloos D, Mol J, Koes R (1996) The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell 85:159–170 Takasaki H, Maruyama K, Kidokoro S, Ito Y, Fujita Y, Shinozaki K, Yamaguchi-Shinozaki K, Nakashima K (2010) The abiotic stress-responsive NAC-type transcription factor OsNAC5 regulates stress-inducible genes and stress tolerance in rice. Mol Genet Genomics 284:173–183 Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50:571–599 Tran LS, Nakashima K, Sakuma Y, Simpson SD, Fujita Y, Maruyama K, Fujita M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Isolation and functional analysis of Arabidopsis stressinducible NAC transcription factors that bind to a droughtresponsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell 16:2481–2498
123
778 Udvardi MK, Kakar K, Wandrey M, Montanari O, Murray J, Andriankaja A, Zhang JY, Benedito V, Hofer JM, Chueng F, Town CD (2007) Legume transcription factors: global regulators of plant development and response to the environment. Plant Physiol 144:538–549 Vilela B, Moreno-Cortes A, Rabissi A, Leung J, Pages M, Lumbreras V (2013) The maize OST1 kinase homolog phosphorylates and regulates the maize SNAC1-type transcription factor. PLoS One 8:e58105 Wu Y, Deng Z, Lai J, Zhang Y, Yang C, Yin B, Zhao Q, Zhang L, Li Y, Xie Q (2009) Dual function of Arabidopsis ATAF1 in abiotic and biotic stress responses. Cell Res 19:1279–1290
123
Plant Cell Rep (2014) 33:767–778 Yanhui C, Xiaoyuan Y, Kun H, Meihua L, Jigang L, Zhaofeng G, Zhiqiang L, Yunfei Z, Xiaoxiao W, Xiaoming Q (2006) The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol Biol 60:107–124 Zheng X, Chen B, Lu G, Han B (2009) Overexpression of a NAC transcription factor enhances rice drought and salt tolerance. Biochem Biophys Res Commun 379:985–989 Zhu J-K (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247
