Introduction

Abiotic stresses, such as drought, high-salinity, low-temperature and oxidative stresses, are principal causes limiting crop productivity throughout the world (Mahajan and Tuteja 2005). Under stressful conditions, the expression of many genes is induced, which encode dehydrins, kinases, phosphatases, transcription factors and detoxification proteins (Brini et al. 2007; Huang et al. 2009; Kim et al. 2012; Li et al. 2012; Zhang et al. 2012). The regulation of these genes leads to physiological and metabolic changes, which facilitate the survival of the plants (Zheng et al. 2009). Transcription factors play an important role in the response to abiotic stresses by regulating the expression of stress-related genes (Ding et al. 2015; Mao et al. 2015; Wang et al. 2015).

NAC(NAM, ATAF1,2 and CUC2) transcription factors belong to a plant-specific transcription factor family that comprises of 117, 151, 152 and 152 predicted members in Arabidopsis, rice, soybean and maize respectively (Shao et al. 2015). NAC proteins contain a conserved DNA-binding NAC domain within the N-terminal and a diverse transcription regulatory domain in the C-terminal (Hu et al. 2010). Previous studies have showed that NAC proteins are involved in many biological processes, such as formation of the apical meristem, flowering, initiation and elongation of lateral roots, construction of cell walls, response to hormones, and fruit ripening, etc. (Kim and Park 2007; Souer et al. 1996; Xie et al. 2000; Zhong et al. 2006; Zhu et al. 2014). Many NAC transcription factors have been found to modulate the response to abiotic stresses and ABA in both Arabidopsis and rice (He et al. 2005; Hu et al. 2008; Wu et al. 2009; Zheng et al. 2009). In Arabidopsis, VNI2 (induced by high-salinity and ABA) is a positive regulator of resistance to salt stress. VNI2 transgenic plants are less susceptible to high-salinity, while mutant seedlings are severely affected by high-salinity (Yang et al. 2011). On the contrary, some salt stress-related NAC TFs play a negative role in salt tolerance. For example, NTL8 encoding a membrane-bound NAC transcription factor, was induced by high-salt, but unaffected by ABA. NTL8 negatively regulates the salt response in germination via the GA pathway (Kim et al. 2008). ANAC092, which is also known as AtNAC2 and ORE1, also negatively regulates salt tolerance and salinity-induced chlorophyll loss (Balazadeh et al. 2010). In rice, some members of NAC TFs positively regulate the response to salt stress, such as SNAC1 (Hu et al. 2006), ONAC045 (Zheng et al. 2009), OsNAC6 (Takasaki et al. 2010), OsNAC5 (Song et al. 2011; Takasaki et al. 2010), OsNAP (Chen et al. 2014), ONAC106 (Sakuraba et al. 2015) and ONAC022 (Hong et al. 2016), etc.

ATAF1 was one of the first NAC proteins identified in Arabidopsis. The expression of ATAF1 was drastically induced by NaCl, drought, ABA and wound treatments (Lu et al. 2007; Wu et al. 2009). Induction of ATAF1 expression indicated that ATAF1 plays a vital role in response to abiotic stresses. Plants in which ATAF1 is abolished displayed sensitivity to drought, but insensitivity to NaCl and ABA, while the overexpressors show reverse phenotypes (Wu et al. 2009). Further study shows that ATAF1 interacts with the catalytic subunits AKIN10 and AKIN11 of SnRK1, which is a key regulator of ABA signaling pathways (Kleinow et al. 2009). ATAF1 also directly regulates the expression of the key abscisic acid biosynthetic gene NCED3 and ABA transport gene ABCG40 (Garapati et al. 2015; Jensen et al. 2013). In addition, ATAF1 directly binds to the promoters of ORE1and GLK1 which encode key chloroplast maintenance and senescence-promoting TFs, respectively, resulting in activation of ORE1 and repression of GLK1 transcription (Garapati et al. 2015). Briefly, ATAF1 regulates abiotic stress tolerance and senescence through the direct regulation of the photosynthesis and senescence transcriptional pathway mediated by ABA and H2O2. Meanwhile, ATAF1 is up-regulated by jasmonic acid but down-regulated by salicylic acid, jasmonic acid, and 1-amino cyclopro-pane-1-carboxylic acid, all of which function in defense of plants against pathogens (Wang et al. 2009; Wu et al. 2009). The overexpression of ATAF1 enhances susceptibility to P. syringae pv. tomato DC3000, B. cinerea, and Alternaria brassicicola (Wang et al. 2009). All of the above evidence implies that ATAF1 is a key point of the crosstalk between biotic and abiotic stress pathways and functions as a switch between plant abiotic stress tolerance and defense (Mauch-Mani and Flors 2009). To study the function of ATAF1 in monocotyledon plants, we generated transgenic rice of ATAF1. The transgenic rice significantly enhanced tolerance to salt and cold, as well as decreased sensitivity to ABA. ATAF1 overexpression in rice elevated the transcription of some stress-related genes, which may account for the salt tolerance of the transgenic rice.

Materials and methods

Plant growth conditions

The seeds were surface sterilized with 70 % ethanol for 1 min and washed two times with sterile water. They were sterilized with 30 % NaClO for 50–60 min, and washed five times with sterile water. The sterile seeds were put on sterile filter paper to dry and then transferred to 1/2 MS medium containing 0.8 % Phytagel. Two-week-old seedlings were harvested for RNA extraction.

Constructs and transformation

The full length of ATAF1 CDS was cloned into pCambia2300 under the ubiquitin promoter and octopine synthetase (OCS) terminal. The expression of NPTII, a selected marker gene, was driven under the control of the CaMV 35S promoter and 35S poly A terminal. The whole expression region was located between left border (LB) and right border (RB) (Fig. 1a). The confirmed construct, pCAMBIA2300-ATAF1, was transformed into Huang Huazhan (Oryza sativa L. subsp. indica) by an Agrobacterium-mediated transformation method to generate transgenic rice.

Fig. 1
figure 1

The expression level of ATAF1 in transgenic plants. a The schematic diagram of the ATAF1 overexpression construct. b Northern blot was performed to check the transcription of ATAF1. Arrows indicated selected lines for further study. c RT-PCR was performed to confirm the expression level of the transgenic lines selected for further study

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RNA isolation and northern blot

Total RNA was isolated using the hot phenol method, and 10 μg was applied in each lane for RNA gel analysis with the α-32p-labeled ATAF1 specific probe to check the transcription of ATAF1 in transgenic rice. For semi-quantitative PCR, DNaseI-treated total RNA (2 μg) was denatured and reverse transcribed using the M-MLV reverse transcriptase at 42 °C for 60 min according to the manufacture’s instruction (Promega). Real time-PCR was performed on the Bio-Rad real-time PCR system using SYBR mixture (Bio-Rad) with the flowing program: 95 °C for 3 s; 40 cycles of 95 °C for 5 s, and 60 °C for 5 s, the plates were then read.

Salt, cold and ABA tolerance assay

For the germination assay, seeds of the transgenic rice and vector controls were germinated in water and in water containing 150 mM NaCl and the number of germinated seeds was counted every day. To study the effect of salt stress on post-germination growth, the seeds were sown on 1/2 MS medium plus 0 or 150 mM NaCl. Two and three weeks later, the root and shoot length respectively, of transgenic rice growing on 1/2 MS medium plus 0 mM or 150 mM NaCl were measured. To study the response to ABA, seeds from the transgenic rice and vector controls were germinated in water containing 0, 4 and 8 μM ABA. After 8 days, the height of the transgenic rice was measured. To check the response of young seedlings to cold stress, seeds were germinated for 2 days at 37 °C in water. The most uniformly germinated seeds were sown into a 96-well plate from which the bottom had been cut off. The plate was floated on Hoagland’s nutrient solution for 4 weeks in a greenhouse. For cold treatment, two-week-old vector control and transgenic seedlings were incubated in a growth chamber at 4 °C for 30 h and then recovered for 5 days.

Results

Identification of transgenic rice by northern blot and RT-PCR

To investigate the functions of ATAF1 in rice, we generated transgenic rice by Agrobacterium-mediated transformation method to overexpress ATAF1. More than ten independent lines were screened with G418 and then transferred to the field for T2 seeds. To check the transcripts of ATAF1, we performed northern blot with an ATAF1-specific probe. Most of the transgenic rice exhibited a high expression level, but similar results were not observed in the vector control plants. Four homozygous lines, including one line for vector control were selected for further studies, named Vector, OX-1, OX-2 and OX-3 (Fig. 1b). To confirm the northern blot results, we amplified ATAF1 using specific primers by RT-PCR. As shown in Fig. 1C, we can detect the transcripts of ATAF1 in transgenic rice, but not in the vector control plants (Fig. 1c).

Overexpression of ATAF1 improved the salt tolerance of transgenic rice

ATAF1 was involved in the response to salt stress in Arabidopsis. The seeds of the transgenic rice and the vector control were germinated in water containing 0 mM and 150 mM NaCl and the number of germinated seeds was counted every day. After 3 days of germination in water containing 150 mM NaCl, only 29 % of the vector control seeds were germinated, whereas more than 47 % of the transgenic seeds germinated rapidly (Fig. 2a). The higher germination rate of the transgenic seeds compared with that of WT under high-salinity condition indicated the superior salt tolerance of ATAF1-overexpressing rice. To test the salt tolerance in post-germination growth, germinated seeds were transferred to 1/2 MS medium plus 150 mM NaCl and 0 mM NaCl as a control, and then grown in a growth chamber (16 h light/8 h dark, 28 °C). Fifteen days later, the root and shoot length of the plants that grew on 1/2 MS medium without NaCl were measured. There was no difference in growth performance between the transgenic and vector control plants. After 3 weeks, these same parameters were measured for plants that grew on 1/2 MS medium containing 150 mM NaCl. We found that not only the root length, but also the shoot length of the transgenic rice (shoot length: 18.57, 18.55, 15.95 cm; root length: 7.60, 9.06, 5.88 cm) was significantly longer than that of the control plants (shoot length: 12.5 cm; root length: 3.78 cm) (Fig. 2b). The overexpression of ATAF1 improved the salt resistance compared with that of the vector control not only in the germination stage but also in the post-germination stage.

Fig. 2
figure 2

Response to salt treatment of ATAF1 transgenic rice. a Growth performance (upper) and germination rate (lower) of transgenic plants and vector control in water containing 150 mM NaCl. Photographs were taken after 8 days. The data represent the mean ± SE, n = 3. b Root length and shoot height of the transgenic and vector control plants on MS medium with 0 mM NaCl for 2 weeks and 150 mM NaCl for 3 weeks. The data represent the mean ± SE, n ≥ 20. Asterisks indicate significant differences (*P ≤ 0.05; **P ≤ 0.01) between the vector control and transgenic plants

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Overexpression of ATAF1 decreased ABA sensitivity

ABA is an important phytohormone and plays an essential role in the response to salt stress. The expression of ATAF1 was clearly induced in Arabidopsis by high-salinity and ABA. Therefore, we investigated the response of transgenic plants to ABA in this study. The height of the ATAF1 overexpression showed no large difference from that of the vector control plants. However, the growth inhibition caused by ABA was reduced in the overexpressor plants. The shoot elongation values of three transgenic lines grown in water containing 4 μM ABA were 2.16, 2.37 and 2.29 cm, respectively, and these values were longer than those of vector control plants (1.72 cm). When the concentration of ABA was increased to 8 μM, the shoot length of the vector control plants decreased to 1.36 cm, which was significantly shorter than that of the transgenic plants (1.90, 2.02 and 1.91 cm, respectively) (Fig. 3a, b). Our results showed that ATAF1 decreased the sensitivity of transgenic rice to ABA.

Fig. 3
figure 3

Response to ABA of ATAF1 transgenic rice. a Growth performance of transgenic plants and vector control in water containing 0, 4 and 8 μM ABA. b Plant height of transgenic rice and vector control after 8 days, bar = 1 cm. The data represent the mean ± SE, n ≥ 52. Asterisks indicate significant differences (**P ≤ 0.01) between the vector control and transgenic plants

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ATAF1 elevated the expression of OsLEA3, OsSalT1 and OsPM1

To understand the mechanism of salt tolerance mediated by ATAF1, real-time PCR was performed to screen some marker genes involved in salt stress using two independent lines OX-1 and OX-2. Three genes were induced significantly after salt treatment in transgenic plants, including OsLEA3, OsSalT1 and OsPM1 (Fig. 4). The expression level in the overexpressor was much higher than that in the vector control plants. Promoters of these genes were analyzed. It was found that promoters of the three genes contained 5, 7 and 6 ATAF1-binding motifs, TT[A,C,G]CGT or T[A,C,G]CGT[A,G] (Jensen et al. 2013), respectively (Table S1), suggesting that ATAF1 might bind to the promoters of OsLEA3, OsSalT1 and OsPM1 to activate the transcription of these genes.

Fig. 4
figure 4

Expression analysis of salt tolerance-associated genes by real-time-PCR. Two-week-old seedlings were treated with 250 mM NaCl for 0, 4 and 12 h. Real-time-PCR was performed using OsLEA3 (a), OsPM1 (b) and OsSalT1 (c) specific primers. Two independent lines and a vector control line were used. The data represent the mean ± SE, n = 3. Asterisks indicate significant differences (*P ≤ 0.05; **P ≤ 0.01) between the vector control and transgenic plants

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Discussion

The overexpression of ATAF1 in Arabidopsis leads to an increased sensitivity to NaCl and ABA but enhances plant tolerance to drought (Wu et al. 2009). In this study, the overexpression of ATAF1 in rice enhances salt and ABA tolerance but has no effect on drought tolerance (Fig. 2). The mechanisms of the response to salt, drought and ABA in Arabidopsis and rice are speculated to be different. Many NAC TFs of Oryza sativa and Zea mays show relatively low homologies with NAC family members from dicot species (Mao et al. 2012). Therefore, the biological functions of homologous NAC TFs in Arabidopsis and rice are not the same absolutely. In rice, OsNAC5 and OsNAC6 belong to the ATAF subfamily, as do ATAF1 and ATAF2 in Arabidopsis (Ohnishi et al. 2005). The expression of OsNAC5 and OsNAC6 are induced by ABA, drought and salt stresses (Ohnishi et al. 2005; Takasaki et al. 2010). The overexpression of both in rice improves salt and drought tolerance (Nakashima et al. 2007; Song et al. 2011; Takasaki et al. 2010). OsNAC5 and OsNAC6 could bind to the promoter of OsLEA3, which is a stress-related gene (Takasaki et al. 2010). In this study, three genes, including OsLEA3, OsSalT1 and OsPM1, are induced at higher expression levels in transgenic plants than in the vector control. OsLEA3 encoding dehydrin is induced by drought, salt and ABA. After salt treatment, an accumulation of OsLEA3 maintains the structure and function of the cell to facilitate its subsequent recovery (Chourey et al. 2003). OsSalT1, which encodes mannose-binding jacaline-like lectin, is induced by various stress treatments, including MS salt, air drying, PEG, NaCl and KCl. OsSalT is responsible for changing the concentration of Na+ to prevent damage and enhance salt tolerance (Claes et al. 1990; Zhang et al. 2000). Wheat WPM1 encodes a hydrophobic polypeptide and is involved in ABA-mediated freezing tolerance (Koike et al. 1997). OsPM1 is a homologue gene of WPM1 and induced by ABA, drought and salt (Zheng et al. 2009). OsLEA3, OsSalT1 or OsPM1 are related to improving the tolerance of rice to abiotic stresses and have been used in several studies as gene markers (Chen et al. 2014; Chourey et al. 2003; Hong et al. 2016; Yang et al. 2012; Zheng et al. 2009). We therefor analyzed the promoters of these three genes and found that there are several ATAF1-binding motifs in promoters of OsLEA3, OsSalT1 and OsPM1, respectively (Table S1). One possibility for the mechanism of improving salt tolerance might be that ATAF1 directly binds to the promoters of OsLEA3, OsSalT1 and OsPM1 and transactivates their expression.

Previous studies show that ectopic expression of stress-related genes significantly improves stress tolerance in crops. Overexpression of ZmCBF3, which encodes a maize C-repeat-binding TF in rice, improves tolerance to drought, high-salt, and low-temperature stresses without growth retardation under normal growth conditions (Xu et al. 2011). TaMnSOD, which encodes manganese superoxide dismutase, was cloned from Tamarix androssowii (Wang et al. 2010). TaMnSOD transgenic cotton plants improve drought tolerance through enhanced development of the root and leaf systems and the regulation of superoxide scavenging, thereby increasing the biomass as well as the root and leaf systems compared with the controls (Zhang et al. 2014). AtCKX1 from Arabidopsis thaliana encoding cytokinin dehydrogenase 1 is ectopically expressed in barley under the control of a mild root-specific beta-glucosidase promoter from maize. Under severe drought stress, all transgenic genotypes maintained higher water contents and showed better growth and yield parameters during revitalization (Pospisilova et al. 2016). Thus, ectopic expression of foreign genes is an effective method to enhance stress resistance in crops by transgenic technology. In this study, the overexpression of ATAF1 improves the salt and cold tolerance (Fig. S1), but reduces the grain yield of rice in the field (Fig. S2b). Moreover, transgenic plants have a shorter plant height than that of the vector control (Fig. S2a). The number of valid tillers and filled grain rate are not affected by overexpression of ATAF1 (Fig. S2c, d). ATAF1 is overexpressed in rice under the control of the ubiquitin promoter which is a constitutive expression promoter. Constantly high expression level in all tissues might explain the growth retardation and low grain yield. The overexpression of many stress-related transcription factors leads to growth retardation, including AtDREB1A, AtDREB2A, OsWRKY89, MusabZIP53 (Kasuga et al. 1999; Qin et al. 2007, 2008; Shekhawat and Ganapathi 2014; Wang et al. 2007). A previous study showed that driving expression using a stress-inducible promoter can improve the stress tolerance without a negative effect on plant growth and development (Kasuga et al. 1999). LIP9, which encodes a low-temperature-induced protein, is induced by cold, drought, high-salinity stresses and ABA (Aguan et al. 1991). The overexpression of OsNAC6 driven by the promoter of LIP9 enhances the tolerance of rice to abiotic stress but does not cause defects in productivity (Nakashima et al. 2007). The ubiquitin promoter can be changed to a stress-induced promoter to increase the yield of transgenic rice. Therefore ATAF1 can serve as a candidate gene to enhance salt tolerance in rice as long as the spatial–temporal expression is controlled.