Plant Physiology and Biochemistry 86 (2015) 34e43

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Research article

Overexpression of wheat NF-YA10 gene regulates the salinity stress response in Arabidopsis thaliana Xiaoyan Ma 1, Xinlei Zhu 1, Chunlong Li, Yinling Song, Wei Zhang, Guangmin Xia, Mei Wang* The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Science, Shandong University, Jinan 250100, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 September 2014 Accepted 15 November 2014 Available online 17 November 2014

The nuclear factor Y (NF-Y) transcription factor is formed by the interaction of three distinct subunits (NF-YA, -YB and -YC). It targets the CCAAT box, a common cis-element in eukaryotic promoters. Here, the bread wheat gene TaNF-YA10-1 has been isolated from the salinity tolerant cultivar SR3. Recombinant TaNF-YA10-1 was heterologously produced in Escherichia coli, and the purified protein successfully bound to the CCAAT motif in vitro. TaNF-YA10-1 was down-regulated by the imposition of salinity and abscisic acid (ABA). The constitutive expression of TaNF-YA10-1 in Arabidopsis thaliana significantly increased the plant's sensitivity to salinity and repressed its sensitivity to ABA as judged from the seed germination, cotyledon greening and the relative root growth. The transcription of stress-related genes AtRAB18, AtRD29B, AtABI5, AtCBF1 and AtCBF3 was downregulated in TaNF-YA10-1 overexpression transgenic plants. The data provide supportive evidence that TaNFYA10-1 is involved in the regulation of growth under salinity stress conditions. © 2014 Published by Elsevier Masson SAS.

Keywords: NF-Y transcription factor CCAAT-box Salinity stress Abscisic acid SR3 Wheat

1. Introduction Soil salinity is among the most severe environmental constraints on plant growth and crop production (Munns and Tester, 2008). It affects plant function as a result of both the toxicity of excessive sodium (and other) ions, and the osmotic stress it imposes on the root. The Arabidopsis thaliana response to salinity stress has been intensively studied, leading to the identification of a number of key pathways; some, but not all, of these are regulated by the phytohormone abscisic acid (ABA) (Zhu, 2001). Activation of the Salt Overly Sensitive (SOS) pathway helps to maintain ionic homeostasis, while both ABA-independent and -dependent signaling pathways regulate an array of genes for response to osmotic stress caused by high salt or other stresses (Munns and Tester, 2008; Wang et al., 2003; Zhu, 2002). Many of the pathways are dependent on the activation of a transcription factor, such as a CBF/DREB, a NAC or a RING-H2 zinc finger protein (Hu et al., 2006; Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Ko et al., 2006).

* Corresponding author. School of Life Sciences, Shandong University, Jinan 250100, PR China. E-mail address: [email protected] (M. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.plaphy.2014.11.011 0981-9428/© 2014 Published by Elsevier Masson SAS.

NF-Y transcription factors, also called CCAAT-binding factor or “heme-activated protein”, are ubiquitous in eukaryotic genomes. It is composed of three subunits, namely NF-YA (¼HAP2), -YB (¼HAP3/CBF-A) and -YC (¼HAP5/CBF-C) (Frontini et al., 2004; Kahle et al., 2005). The NF-YB-YC heterodimer is suggested to be assembled in the cytoplasm, which subsequently translocates into the nucleus and interacts with NF-YA to form an active heterotrimer (Frontini et al., 2004; Steidl et al., 2004). This complex has high affinity and sequence specificity for the CCAAT box, which is a ciselement that exists in approximately 25% of eukaryotic gene promoters (Cai et al., 2007; Ceribelli et al., 2008; Maity and de Crombrugghe, 1998). The NF-YA subunit confers the sequence specificity to the complex via an unknown mechanism (Dolfini et al., 2012; Mantovani et al., 1994). In contrast to animal and yeast NF-Y genes, which are single copy, plant homologs are typically present in the form of multigene families (Edwards et al., 1998). In A. thaliana, for example, there are, respectively 10, 13 and 13 copies of NF-YA, NF-YB and NF-YC (Siefers et al., 2009), while in the model grass species Brachypodium distachyon and in bread wheat (Triticum aestivum), the number of NF-Y genes represented is, respectively, 36 and 35 (Cao et al., 2011; Stephenson et al., 2007). Due to the potential combinatorial diversity among numerous NF-Y factors, as well as the high affinity

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and sequence specificity for the extensive CCAAT box in the eukaryotic genomes, these members are involved in the regulation of various developmental processes and stress responses. It has been demonstrated that AtNF-YB9 (LEC1) plays a pivotal role in embryo development (Lee et al., 2003a). Altered expression of AtNF-YB1 and AtNF-YB2 could greatly affect the flowering time (Cai et al., 2007; Chen et al., 2007; Wenkel et al., 2006). Some NF-YA subunits such as AtNF-YA2, AtNF-YA3 and AtNF-YA5 were reported to participate in nitrogen nutrition (Laloum et al., 2013). Moreover, NF-Ys have been identified as regulators of drought tolerance in different plant species. The over-expression of AtNFYA5 and AtNF-YB1 in A. thaliana, and of ZmNF-YB2 in maize, improves plant performance and survival under drought conditions (Li et al., 2008; Nelson et al., 2007). Broader functions of NF-Y factors like regulation of light signaling, ER stress, chloroplast biogenesis etc were also revealed (Laloum et al., 2013; Petroni et al., 2012). As yet, the biological roles of most of the NF-Y family members in the salinity stress response are poorly understood. The salinity tolerant bread wheat cultivar Shanrong No. 3 (SR3) is a derivative of an asymmetric somatic hybrid between the bread wheat cultivar Jinan 177 (JN177) and tall wheatgrass (Thinopyrum ponticum) (Wang et al., 2003). It out-yields JN177 in salinityaffected soil, and it has been planted commercially in China (http://www.seedsd.com/news/news_view.asp?id¼511) (Xia, 2009). Here, it is shown that in the SR3 plant, TaNF-YA10-1 was down-regulated by a number of abiotic stress factors, including salinity. The constitutive expression of TaNF-YA10-1 in A. thaliana resulted in a marked increase in sensitivity to salinity stress and a decrease in sensitivity to ABA, as well as an altered transcription profile with respect to a number of known stress response genes. The data contribute to the elucidation of the role of NF-YA subunits in the salinity stress response.

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sequences were aligned using ClustalW as implemented by Molecular Evolutionary Genetics Analysis (MEGA) software, version 5.0 (Tamura et al., 2011). A phylogenetic analysis was conducted using the neighbor-joining method, applying the following parameters: bootstrap replicates: 1000, Poisson mode and pairwise deletion. 2.3. Reverse transcription (RT)-PCR and quantitative (Q)-PCR analysis Total RNA was isolated from both A. thaliana and wheat seedlings using the TRIzol reagent (Invitrogen), then treated with RNase-free DNase I (Promega). RT-PCR was conducted following the supplier's protocol (Takara). Each qPCR was repeated for three biological replicates, and the reactions were based on FastStart Universal SYBR Green Master (Roche). The reference sequences comprised segments of the wheat actin (AB181991) or the A. thaliana actin (GI:145338402) genes. Primer sequences for RTPCR and qPCR were designed using PRIMER PREMIER 5.0, and are listed in Supplementary Table 1. 2.4. Subcellular localization of TaNF-YA10-1

2. Materials and methods

To generate the p35S::TaNF-YA10-1-GFP plasmid, the TaNF-YA101 open reading frame was amplified by PCR using the primer pair NF-YA10-1GF/R (Supplementary Table 1). The amplicon was inserted into the BamHI/SalI cloning site of a modified CaMV35S-GFP vector (Liu et al., 2014). The plasmids CaMV35S-GFP and CaMV35S:TaNF-YA10-1-GFP were introduced separately into both onion epidermal cells (Lee et al., 2003b) and wheat cv. Yangmai11 protoplasts of suspension cell lines from embryogenic calli (Sheen, 2001). After a 16 h incubation at 23  C in the dark, the GFP signal and chlorophyll autofluorescence were detected by confocal laserscanning microscopy (LSM700; Carl Zeiss), using excitation wavelengths of 488 nm and 647 nm, respectively.

2.1. Plant materials and treatments

2.5. Electrophoretic mobility shift assay

Grain of SR3 and JN177 were germinated on moist filter paper at 20  C for two days. Uniform seedlings were raised in half-strength Hoagland's liquid medium under a 16 h photoperiod at 22  C. Plants were subjected to abiotic stress by adding one of 18% (w/v) PEG6000, 200 mM NaCl, 100 mM H2O2 or 100 mM ABA to the culture solution after two weeks, and seedlings were harvested after exposure to the stress agent for either 1, 3, 12 or 24 h. A. thaliana ecotype Col-0 was used as the transformation target for either pSTART::TaNF-YA10-1 or an empty pSTART vector, using the floral dip method (Clough and Bent, 1998). Progeny of the primary transgenics were germinated in the presence of kanamycin and the inheritance of the transgene in surviving seedlings was confirmed using both genomic PCR and RT-PCR. Two homozygous T4 selections were retained (OE 3e6 and 5e7) for functional analysis. A. thaliana plants were raised under a 14 h photoperiod, a light intensity of 120 mmol m2 s1, a day/night temperature of 22/18  C and a relative humidity of 70%.

To express and purify TaNF-YA10-1 for electrophoretic mobility shift assay, the full-length coding regions of TaNF-YA10-1 was PCR amplified with the primer pairs TaNF-YA10-1-GF and TaNF-YA10-1GR (Supplementary Table 1) and cloned into pET-30a vector. The recombinant plasmids were transformed into Escherichia coli strain BL21 (DE3), and the subsequently expressed fusion protein was purified from homogenised bacterial cells using a His-tagged protein purification kit (R&D Systems) according to the manufacturer's protocol. A DIG Gel Shift kit (Roche) was used to detect DNAprotein interaction. Digoxigenin (DIG) was labeled at the 30 end of the double-stranded oligonucleotides. DIG labeled (0.4 ng/ml), unlabeled (0.1 mg/ml) or mutated CCAAT oligonucleotides (0.1 mg/ml) was mixed with TaNF-YA10-1-His protein or a crude protein extracts of wheat in binding buffer and incubated at room temperature for 15 min. The reaction products were fractionated through a 7.5% non-denaturing polyacrylamide gel, then electrophoretically transferred to a positively charged nylon membrane (Roche) by applying a constant current of 50 mA for 4 h. The DNA was crosslinked to the membrane using a UV stratalinker (Stratagene). The membrane was blocked for 1 h at room temperature in 1 blocking reagent, then challenged with a 1:20,000 dilution of anti-DIG antibody coupled to alkaline phosphatase for 30 min. The membrane was rinsed twice in 0.1 M maleic acid (pH 7.5), 0.15 M NaCl, 0.3% (v/v) Tween 20 for 20 min, then after a 5 min equilibration in 100 mM TriseHCl (pH 9.5), 100 mM NaCl, a 1:100 dilution of the substrate CSPD in the same buffer was added. After a 10 min incubation at 37  C, the membrane was exposed to an X-ray film for 20 min to capture the chemiluminescent signal.

2.2. Gene structure and phylogeny The fragment of the NF-YA homolog identified via microarray analysis (Liu et al. 2012) was used as a BLASTN query against the wheat (T. aestivum) EST database held at the National Center for Biotechnology Information. All matching ESTs were assembled using CAP3 software, and a pair of gene-specific primers designed from this assembly (Supplementary Table 1) was used to amplify a full-length cDNA from a cv SR3 cDNA library. Gene structure was predicted using SMART software (Schultz et al., 1998), and peptide

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2.6. Salt stress and ABA treatment Transgenic and non-transgenic A. thaliana seed were surfacesterilized by immersion in 75% ethanol for 3 min, followed by 95% ethanol for 1 min, air-dried on sterile paper and plated on 0.7% w/v agar containing half strength Murashige and Skoog (MS) medium. The plates were held at 4  C in the dark for three days, then transferred to a chamber supplying a 16 h photoperiod of 100 mmol m2 s1 light, 22 ± 1  C (day), 16 ± 4  C (night) and ~70% relative humidity. For measuring root growth after NaCl treatment, four-day-old seedlings grown on vertically orientated agar plates were transferred to a half strength MS agar plate supplemented with either 0 or 150 mM NaCl. Primary root growth was assessed after eight days. To quantify survival under salinity stress, soil-grown seedlings were watered normally for three weeks, then watered with 250 mM NaCl, and watered with the same salinity solution again six days later. For ABA treatment, the germination assay was performed using approximately 60 surface-sterilized seeds placed on one-half MS medium supplemented with 0, or 1 mM ABA. The seeds were put at 4  C for 3 days before transferring to 22  C for germination. The percentage of seed germination (defined by the emergence of the radicle) was scored over time. For growth measurements, 4day-old seedlings from vertical agar plates were transferred to onehalf MS agar plates supplemented with 0, 75 or 100 mM ABA, increases in primary root length were measured nineteen days later. 3. Results 3.1. TaNF-YA10-1 is down-regulated in wheat by various abiotic stresses Analysis of the microarray-based transcriptomes of SR3 and JN177 seedlings challenged with 200 mM NaCl identified a

sequence which was differentially transcribed in the root and was more abundant in SR3 than in JN177. The sequence matched NF-YA genes and was therefore denoted TaNF-YA10-1. Quantitative real time PCR (qPCR) analysis confirmed that its transcript abundance fell significantly during the course of a plant's exposure to 200 mM NaCl, and more so in SR3 than in JN177 (Fig. 1A). High salinity often stimulates the production of ROS and ABA (Mittler et al., 2011). Here, exposure to either 100 mM ABA or 100 mM H2O2 also induced a decrease in transcript abundance (Fig. 1B and Supplemental Fig. 1). High salinity also consists of osmotic stress (Munns and Tester, 2008). When the plants were challenged by the drought simulator PEG6000, TaNF-YA10-1 transcription was initially declined, but eventually increased (Fig. 1C). TaNF-YA10-1 transcription was detectable throughout the plant, but was strongest in the leaf, root and stem (Fig. 1D). Thus, TaNF-YA10-1 is clearly a stress-responsive gene, and its reduced level of transcription after stress treatment implies that it has a negative role in stress tolerance. 3.2. TaNF-YA10-1 encodes a CCAAT-binding transcription factor Sequence analysis indicated that the full-length 1272 bp TaNFYA10-1 cDNA consists of a 222 bp 50 UTR, a 765 bp open reading frame and a 285 bp 30 UTR. It encodes a 254 residue protein, and an alignment between its genomic and the cDNA sequences showed that the coding sequence is split into five exons (Fig. 2A). A phylogenetic analysis of the gene product's peptide sequence demonstrated a high level of identity with both the Triticum urartu (donor of the bread wheat A genome) NF-YA5 sequence (97% identity) and the Aegilops tauschii (D genome donor) NF-YA5 sequence (95% identity), suggesting that it may be from the A genome of bread wheat (T. aestivum, AABBDD). The sequence also shares homology with the B. distachyon YA-1-like protein (74%), rice

Fig. 1. TaNF-YA10-1 transcript abundance in seedlings at the three leaf stage in response to abiotic stress, as measured by qPCR. SR3 and JN177 roots subjected to 200 mM NaCl (A), 100 mM ABA (B) and 18% PEG (C). (D) Variation in space for transcription. Relative expression levels were normalized with the gene expression of wheat actin (AB181991) in (A) to (C). The values represent the mean of three technical replicates and the experiment was repeated once with similar results, error bars indicate the SD.

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Fig. 2. Structure and phylogeny of TaNF-YA10-1. (A) Gene structure. Exons shown as black boxes, and the introns as black bars. (B) Phylogenetic analysis. The numbers shown at each nodes represent bootstrap values. The bar at the bottom indicates the relative divergence. Zm, Zea mays; Os, Oryza sativa; Ta, Triticum aestivum; Tm, Triticum monococcum; Hv, barley; Bd, Brachypodium distachyon; Si, Setaria italica; Sb, Sorghum bicolor; Gm, Glycine max; Mt, Medicago truncatula; and At, Arabidopsis thaliana.

OsNF-YA (EEE57897.1, 68%), maize NF-YA3 (62%) and wheat WHAP6 (54%) (Fig. 2B). It has retained the conserved residues which define the NF-YA interaction with NF-YB-NF-YC, along with the CCAAT binding sites of the OYE family (Supplemental Fig. 2).

wheat suspension cell culture (Fig. 3B). These results indicate that TaNF-YA10-1 is located in both the nucleus and the cytoplasm.

3.3. Subcelluar localization of TaNF-YA10-1

The EMSA (electrophoretic mobility shift assay) technique was used to detect the interaction between TaNF-YA10-1 and the CCAAT box sequence. Although NF-YA is the subunit that makes sequence-specific contact with CCAAT boxes (Xing et al., 1993), NF-YB and NF-YC have been shown in vitro to be also essential for binding (Maity and De Crombrugghe, 1996; Sinha et al., 1996). Thus, a total protein extract of the wheat leaves were added to the reaction buffer. Weak CCAAT binding activity was detected when the wheat total protein extract was used. However, the binding was significantly enhanced when TaNF-YA10-His protein was

In mammals, the NF-YA factors are located in the nucleus and interact with the NFYB/NFYC dimers transported from the cytoplasm (Kahle et al., 2005). Here three nuclear localization sequences were identified in TaNF-YA10-1 (Supplemental Fig. 2). When the CaMV35S::TaNF-YA10-1-GFP transgene was transiently expressed in onion epidermal cells, GFP was detectable in both the cytoplasm and the nucleus (Fig. 3A) and a similar distribution prevailed in transiently transformed protoplasts prepared from a

3.4. TaNF-YA10-1 binds to CCAAT

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Fig. 3. Subcellular localization of TaNF-YA10-1. Transient expression of TaNF-YA10-1-GFP in onion epidermal cells (A), and wheat suspension cell protoplasts (B). GFP only vector driven by the CaMV35S promoter was used as the GFP control. Images were captured by a laser scanning confocal microscope using the following wavelengths: GFP (excitation, 488 nm; emission, 509 nm), and chlorophyll autofluorescence (excitation, 448 nm; emission, 647 nm). Bars ¼ 10 mm. The experiment was repeated three times with similar results.

added with the total wheat protein extract (Fig. 4A). When either a mutated oligonucleotide probe or an unlabeled probe was included with protein, binding intensity was significantly reduced (Fig. 4B). The evidence is therefore that, at least in vitro, TaNFYA10-1 binds to CCAAT.

3.5. The constitutive expression of TaNF-YA10-1 increases the salinity sensitivity of A. thaliana To investigate whether TaNF-YA10-1 works in abiotic stress responses, we constitutively expressed TaNF-YA10-1 in A. thaliana

Fig. 4. DNA binding activity of TaNF-YA10-1-His6 recombinant protein. (A) Heterologously expressed TaNF-YA10-1-His6 in E. coli. The arrow indicated the TaNF10-1-His6 protein. (B) DNA binding activity of TaNF-YA10-1-His6 recombinant protein. Crude protein refers to a total protein extract from wheat leaf. The experiment was repeated three times with similar results.

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(Fig. 5A). Because several independent transgenic lines behaved in a similar manner (data not shown), we performed detailed analyses on two independent overexpression (OE) transgenic lines 3e6 and 5e7. Our data demonstrated that TaNF-YA10-1 gene was downregulated by salt (Fig. 1A), therefore, the TaNF-YA10-1 OE plants could be expected to have less tolerance to salt. To examine this possibility, root growth was evaluated first using the wild type, vector control (VC) line, and TaNF-YA10-1 OE plants. The response of the root to salinity stress was tested by exposing seedlings to 150 mM NaCl. The outcome of the experiment was that the growth of the TaNF-YA10-1 transgenic plants' roots was more constrained than those of the control plants (Fig. 5B and C). For soil-grown plants, it was clear that

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the two transgenic lines were less able to tolerate the salinity stress than the controls (Fig. 5DeH). The fresh weight of the two transgenic lines was, respectively 45.3% and 47.5% that of the VC plants after 14 days of exposure (Fig. 5I). The implication is that TaNF-YA10-1 acts to negatively regulate the response to salinity stress. 3.6. The constitutive expression of TaNF-YA10-1 reduces the ABA sensitivity of A. thaliana The altered salinity sensitivity in TaNF-YA10-1 OE plants led us to investigate whether over-expression of TaNF-YA10-1 also affect sensitivity of plants to ABA. To test this possibility, germination and

Fig. 5. The constitutive expression of TaNF-YA10-1 in A. thaliana increased the sensitivity of transgenic plants to salinity stress. (A) RT-PCR profiles of A. thaliana transgenic lines (3e6 and 5e7), wild type (Col-0) and the empty vector control (VC). (B) and (C) Comparison of root length between Col-0, VC and TaNF-YA10-1 overexpression lines under normal conditions and 150 mM NaCl. The phenotype of three week old VC, Col-0, 3e6 and 5e7 after exposure to 250 mM NaCl for 0 d (D), 4 d (E), 7 d (F), 11 d (G), 14 d (H). (I) Fresh weight (FW) of 3-week-old VC, Col-0, 3e6 and 5e7 plants after a 14 d exposure to 250 mM NaCl. FW of individual plants between control and TaNF-YA10-1 overexpression lines was compared. All data are given as mean ± SD from three independent experiments. * represents a statistically significant (P < 0.05) difference, as determined by the Student's t-test.

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early seedling growth of the wild type, VC and the TaNF-YA10-1 OE plants were observed on 1/2 MS medium without ABA, or supplemented with different concentrations of ABA. As shown in Fig. 6A, seed germination of TaNF-YA10-1 OE was less severely inhibited by 1 mM ABA application than that of VC and the wild type at 24 h and 48 h. The same trend was also observed for cotyledon greening and expansion in the presence of 1 mM ABA (Fig. 6B). As TaNF-YA10-1 OE showed decreased sensitivity to ABA in seed germination and cotyledon greening, to gain further information, the root elongation of the seedlings grown under medium containing a higher gradient concentration of ABA was evaluated. Four-day-old MS-grown seedlings were transferred to fresh medium supplemented with 75 and 100 mM ABA. After 19 days, growth of both the TaNF-YA10-1 OE and VC plants was much reduced by ABA compared to their untreated controls. However, ABA inhibited seedling growth was much less severe in TaNF-YA10-1 OE than that in the VC plants, and TaNF-YA10-1 OE showed longer primary roots and more lateral roots than VC plants when exposure to 75 or 100 mM ABA (Fig. 6C, D and Supplemental Fig. 3). These results indicate that constitutive expression of TaNF-YA10-1 reduced ABA sensitivity in A. thaliana. 3.7. Constitutive expression of TaNF-YA10-1 down-regulates a number of stress-responsive genes To explore how the TaNF-YA10-1 gene negatively regulate plant responses to salt stress, we performed qPCR to identify salt-

regulated genes that are differentially expressed in the TaNFYA10-1 OE transgenic plants. The ABA-dependent genes AtRAB18, AtRD29B and AtABI5 were strongly induced by NaCl in both TaNFYA10-1 OE transgenic plants and control plants. However, their expression levels were much lower in the TaNF-YA10-1 OE transgenic plants than in the Col-0 and VC (Fig. 7). To determine whether its role was limited to the ABA-regulated pathway, expression levels of several ABA-independent genes were also assayed. Among them, followed NaCl treatment the transcript levels of AtCBF1 and AtCBF3 were increased by 25.5- and 30.1-, 22.7- and 23.2- fold for the WT and VC, 11.6- and 16.1-, 6.9- and 9.9-fold for transgenic plants 3e6, 5e7, respectively. Therefore, NaCl can also strongly induce the expression of above two genes and their transcript levels were much lower in the TaNF-YA10-1 OE transgenic plants than in the wild type and VC (Fig. 7). 4. Discussion Nuclear factor Y is member of a large plant transcription factor family. A number of NF-Y proteins are known to act as regulators of the drought response: these include AtNF-YA5, which is also responsive to ABA (Li et al., 2008), and AtNF-YB1, ZmNF-YB2, and TaNF-YB2, which have all been reported to be involved in determining drought tolerance (Nelson et al., 2007; Stephenson et al., 2007). However, reports of NF-YAs in salinity stress are still sparse. In this study, by physiological analysis of Arabidopsis

Fig. 6. The constitutive expression of TaNF-YA10-1 in A. thaliana reduced the sensitivity of transgenic plants to ABA. (A) Germination rate of Col-0, VC and TaNF-YA10-1 OE lines (3e6 and 5e7) at indicated time under 1 mM ABA. (B) Green cotyledons between Col-0, VC, 3e6 and 5e7 exposed to 1 mM ABA for 10 days. (C) Root phenotype of transgenic A. thaliana lines compared with control plants under normal conditions, 75 and 100 mM ABA for 19 days. (D) Total root length of above seedlings. The VC plants are a transgenic line carrying an empty vector. Col-0 is the wild type. Bars ¼ 1 cm in (C). All data are given as mean ± SD from three independent experiments. The asterisks and double asterisks represent significant difference determined by the Student's t-test at P < 0.05 and P < 0.01 respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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transgenic plants constitutively expressing TaNF-YA10-1, the possible role of TaNF-YA10-1 in plant salt stress was explored. TaNF-YA10-1 was down-regulated by a number of different abiotic stress agents (Fig. 1, Supplementary Fig. 1), as are at least eight TaNF-Y genes (Stephenson et al., 2007). However, many of the AtNF-Ys are known to be up-regulated by stress. For example, At-NFYA5 is strongly induced by drought, as well as by osmotic and salinity stress (Li et al., 2008). At-NF-YC2 is strongly induced in response to number of abiotic stresses, including drought. AtNF-YB2 is also responsive to multiple abiotic stress (Cai et al., 2007). So far, no AtNFY genes have been reported to be down-regulated (Hackenberg et al., 2012) in the way that TaNF-YA10-1 was. This may reflect functional divergence between monocotyledonous and dicotyledonous species. Furthermore, the transcription abundance of TaNF-YA10-1 is higher in salinity tolerant asymmetric somatic hybrid SR3 than that in its parent salinity sensitive bread wheat JN177 under either control conditions or abiotic stresses (Fig. 1A). This seems to be inconsistent with the result that when expressed heterologously in A. thaliana, it has a marked negative effect on the plant's salinity response (Fig. 5). To be noted, a set of 226 genes which were differentially expressed (by at least two fold) in SR3 seedlings under stress (200 mM NaCl or 18% PEG) was identified by microarray

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analysis (Liu et al., 2012). Thus, many other genes controlling the salt stress response may contribute more to the stress response of SR3. For example, we have indicated that TaOPR1 (Dong et al., 2013) and wheat Allene Oxide Cyclase (Zhao et al., 2014) play an important role in SR3 salinity stress. More importantly, we proved that TaSRO1 located in chromosome 5 may be the major stress tolerance locus in SR3, genetic and biochemical analysis showed that Tasro1 was largely responsible for the stress tolerance of SR3 (Liu et al., 2014). Thus, it is possible that the stress tolerance of SR3 does not result from the expression of TaNF-YA10-1 but some other genes, although TaNF-YA10-1 plays a role in salinity stress response. The presence of the conserved CCAAT-binding motif, subcellular localization of TaNF-YA10-1 protein and its demonstrated DNAbinding activity are all consistent with TaNF-YA10-1 representing a functional NF-YA subunit (Fig. 3, 4 and Supplemental Fig. 2). The NF-YA proteins have a well established role in the plant stress response, and by implication, in the plant's means to cope with stress. AtNF-YA1, for example, regulates post-germination growth arrest when the plant senses salinity stress (Li et al., 2013). In this study, the constitutive expression of TaNF-YA10-1 in A. thaliana lowered the level of salinity tolerance and ABA sensitivity as judged from the seed germination, cotyledon greening and the relative

Fig. 7. Relative transcript levels in 2-week-old seedlings of genes involved in salinity stress response pathway in A. thaliana. qPCR profiles of stress-responsive genes in VC, Col-0 and TaNF-YA10-1 overexpression lines under non-stressed conditions and 200 mM NaCl. The relative expression levels of VC under normal conditions (control) were set at 1.0. The VC plants are a transgenic line carrying an empty vector. Col-0 is the wild type. 3e6 and 5e7 are transgenic A. thaliana lines constitutively expressing TaNF-YA10-1 cDNA from SR3. Error bars represent mean ± SD from three technical replicates. The experiment was repeated once with similar results.

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root growth under salt stress and ABA treatment (Figs. 5 and 6). It is consistent with the case that TaNF-YA10-1 was down-regulated when exposed to salinity and ABA (Fig. 1A and B). The response to both salinity and drought stress can be modulated by either an ABA-dependent or an ABA-independent pathway. In transgenic A. thaliana constitutively expressing TaNF-YA10-1, the ABAdependent pathway genes AtRAB18, AtRD29B and AtABI5, as well as the ABA-independent pathway genes AtCBF1 and AtCBF3, were all down-regulated, whether or not the plants were exposed to salinity stress (Fig. 7). All of these genes are known to be inducible by salinity stress, and it is assumed that their expression contributes towards salinity tolerance (Apse et al., 1999; YamaguchiShinozaki and Shinozaki, 2006). These results imply that stress adaptation in the TaNF-YA10-1 producing transgenic A. thaliana was complex, involving both ABA-dependent and -independent pathways. It is an effective way to study the function and possible signaling pathways of wheat genes in A. thaliana, however, further research should be conducted to prove that the constitutive expression of TaNF-YA10-1 in wheat showed consistent phenotypes with A. thaliana TaNF-YA10-1 OE transgenic plants. Certain NF-Y subunits have been reported to act pleiotropically in the course of plant growth, development and the response to abiotic stress. An example is the gene AtNF-YA1, which is active during male gametogenesis, embryogenesis, seed development and the salinity stress response (Li et al., 2013; Mu et al., 2013). Here, besides the salinity sensitive phenotype, the 35S::TaNF-YA101 plants exhibited a late flowering phenotype compared with that of wild type and VC A. thaliana (data not shown). The distinct functions carried out by TaNF-YA10-1 may be explicable in terms of the diversity of interactions made possible by the capacity of NFYA10-1 to interact with a range of NF-YB/NF-YC factors. It is interesting to gain insight into the connection of distinct TaNFYA10-1 functions, and further study on proteineprotein interactions will also be needed to investigate the inner mechanisms. Accession numbers The GenBank accession numbers for the genes used in this article are as follows: TaNF-YA10-1 (KM983028), NF-YA5 [Triticum urartu] (EMS62064), NF-YA5 [Aegilops tauschii] (EMT02956), BdNFYA1-like (XP_003570525), SiNF-YA8-like (XP_004954092), OsNF-YA (EEE57897), HvNF-YA (BAJ99575), SbNF-YA (XP_002454647), ZmNFYA3 (AFW73715), TaWHAP6 (AAS78480), TaWHAP8 (AAS78482), TmNF-YA9 (ADY38378), TmNF-YA7 (ADY38377), AtNF-YA5 (NP_175818), AtNF-YA3 (NP_565049), AtNF-YA8 (NP_173202), AtNFYA6 (NP_188018), OsNF-YA1 (ABF94257), OsNF-YA2 (AAW39026), GmNF-YA1 (ACV44452), MtNF-YA8 (JQ918273), GmNF-YA3-like (XP_006597204) and MtNF-YA4 (JQ918269). Author contributions XY Ma and XL Zhu performed the experiments; CL Li and YL Song characterized transgenic plants; W Zhang and GM Xia revised the manuscript; M Wang designed the experiments and wrote the manuscript. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledges This research was financially supported by grants from the National Natural Science Foundation of China (31471158), the Shandong Province Scientific Research Award Foundation for Excellent

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