GmNAC15 overexpression in hairy roots enhances salt tolerance in soybean

LI Ming, HU Zheng, JIANG Qi-yan, SUN Xian-jun, GUO Yuan, QI Jun-cang ZHANG Hui

1 Agricultural College, Shihezi University, Shihezi 832003, P.R.China

2 National Key Facilities for Crop Genetic Resources and Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

1.Introduction

Soybean is an important commercial crop and is the primary source of oil and protein for humans.China is one of the largest soybean producers in the world, with more than 1 600 000 tons of soybean harvested from more than 300 000 ha each year.However, abiotic stressors, such as salinity, drought, and low temperatures, pose a serious threat to soybean development and yield, especially high salt levels.High salt concentrations seriously limit the production of soybean through negative effects on the cellular, organ,and whole-plant levels (Phang et al.2008).Salt stress is a complicated process that causes water deficits.A lack of water leads to the accumulation of reactive oxygen species(ROS) such as hydrogen peroxide (H2O2) and superoxideHigh concentrations of cytotoxic oxygen can disturb normal cell metabolism through the destruction of the cell membrane structure, which ultimately leads to cell death(Parida et al.2005).

In plants, NAC is a large gene family.NAC comprises three subfamilies: no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC).Several NAC family members in model plants have been identified and characterized, such as 151 in rice (Oryza sativa), 117 in Arabidopsis (Nuruzzaman et al.2010), and 152 in tobacco (Nicotiana tabacum)(Rushton et al.2008).In soybean, 152 NAC genes have been identified (Le et al.2011).The NAC protein consists of two parts: highly conserved N-terminal NAC-binding domain (DB) (150-160 amino acids) and highly variable C-terminal transcription regulatory (TR) region (Puranik et al.2012).The N-terminal plays a role in protein-binding and dimerization, and the C-terminal is responsible for activator or repressor function.The N-terminal of NAC contains five motifs (A-E).A participates in dimer formation, B and E may be involved in different functions of NAC, and C and D are positively charged and responsible for DNA recombination(Ernst et al.2004; Jensen et al.2010).

Many members of the NAC family are relevant to plant development (Feng et al.2014).GmNAC20 promotes lateral root formation by regulating auxin-related genes (Hao et al.2011).CUC1 of Arabidopsis regulates the formation of the shoot apical meristem (Takada et al.2001).CUC1, CUC2,and CUC3 make significant contributions to postembryonic development (Hibara et al.2006).In addition, the NAC family plays important roles in response to abiotic stressors such as salt, drought, and cold.ANAC002, ANAC019, ANAC055,and ANAC72 enhance the drought tolerance of Arabidopsis(Huang et al.2016).Similarly, overexpression of SINAC1,TaNAC29, and ONAC045 in tomato, Arabidopsis, and rice facilitates increased resistance to salt stress (Zheng et al.2009; Yang et al.2011; Huang et al.2015).

The root is the first plant organ damaged by salt in the soil,so it is useful for studying the salt tolerance of plants.The transgenic soybean has been cultivated on a large scale in the world; however, there are many unsolved problems in the Agrobacterium-mediated transformation system, such as the low transformation efficiency and complex procedure.In addition, this method is susceptible to pollution and is time-consuming.These problems can be solved by using Agrobacterium rhizogenes, which infects wound sites and transfers T-DNA from the bacterial cell to the plant cell.K599, one strain of the A.rhizogenes, can be easily injected into the cotyledonary node and effectively induce hairy roots at the infection site to form composite soybean plants that comprise both wild-type shoots and transgenic hairy roots.The A.rhizogenes-mediated transformation system is fast,simple, and highly efficient (Estrada-Navarrete et al.2006;Kereszt et al.2007).In this study, we elucidated the role of GmNAC15 in salt tolerance using the A.rhizogenes-mediated hairy root system.

2.Materials and methods

2.1.Plant materials, growth conditions, and treatments

The soybean cultivar Williams 82 was used to isolate the GmNAC15 gene and to analyze tissue-specific expression.RNA was isolated from the roots, stems, leaves, and cotyledons of 20-day-old seedlings and flowers from mature plants.Seeds were germinated in pots containing vermiculite, and 20-day-old seedlings were subjected to salt, dehydration, abscisic acid (ABA), and cold treatment.The roots of the seedlings were immersed in Hoagland solutions containing 250 mmol L-1NaCl, 20% polyethylene glycol (PEG), and 100 μmol L-1ABA for different periods of time.For the cold treatment, the seedlings were maintained at 4°C for the indicated time period.

2.2.Construction of plant expression vector

Total RNA was isolated from Williams 82 using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA).Genomic DNA was removed with DNaseI (Thermo Scientific, Vilnius,Lithuania) according to the manufacturer’s instructions.First-strand complementary DNA (cDNA) synthesis was performed with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific).The full-length cDNA of GmNAC15 was obtained by PCR with Pfu DNA polymerase (TransGen Biotech, Beijing, China) and a pair of primers (forward primer:5′-ATGAGCAACATAAGCATGGTA-3′; reverse primer:5′-TCAGTAATTGTTCCACATGTG-3′).The amplified products were purified and cloned into the Blunt vector with pEASY-Blunt Simple Cloning Kit (TransGen Biotech).GmNAC15 was cloned into pCAMBIA3301 with the Quick-Fusion Cloning Kit (TransGen Biotech) and a pair of primers(forward primer: 5′-GAACACGGGGGACTCTTGACA TGAGCAACATAAGCATGGTA-3′; reverse primer:5′-TTACCCTCAGATCTACCATGTCAGTAATTGTTCCACA TGTG-3′).

2.3.Agrobacterium rhizogenes-mediated transformation and salt treatment

The seeds were placed into identical pots containing mixed soil (humus:vermiculite=2:1) at a depth of 1-2 cm.Then, the potted seeds were placed in a greenhouse at 28°C and watered daily.Six-day-old seedlings with folded cotyledons were infected by A.rhizogenes K599 with the vector control (pCAMBIA3301) and overexpression vector around the cotyledonary node area using a syringe needle.The soybean plants were maintained under a 12 h light/12 h dark cycle at 28°C.After the initiation of hairy root formation from the infection site, the hairy roots were covered with vermiculite to maintain high humidity.A month later, once the hairy roots were 5-10 cm in length and could support the growth of the plant, the main roots were removed.The plants with hairy roots were transferred into mixed soil(humus:vermiculite=2:1) and watered every 3 days.The hairy roots and leaves were used to quantify the expression levels of GmNAC15.After two weeks, when the hairy roots had restored the health of the plants, the overexpression and control seedlings were irrigated with 250 mmol L-1NaCl solution.Two days later, the leaves and hairy roots were collected to analyze the function of GmNAC15.

2.4.Quantitative reverse transcription PCR

cDNA samples were obtained as described above.qRTPCR was performed with the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific), and the amplification was performed with the Eco Real-Time PCR System(Illumina, San Diego, CA, USA).All reactions were repeated three times.After PCR, the data were quantified using the comparative CTmethod (2-ΔΔCTmethod) based on CTvalues(Livak and Schmittgen 2001).The gene-specific primers for GmNAC15 were 5′-TGGCACACAATGATTCCCT-3′ and 5′-AGCCCTGTTGCATACTTACGG-3′.cDNA samples from the hairy roots of the vector control (VC) and overexpression composite (OE) plants were used to examine the expression of two salt stress-related genes (GmERF3 and GmWRKY54)by qRT-PCR.The corresponding specific primers were sense 5′-CTTGGACGTTGACTTCGAGGCTGAT-3′ and antisense 5′-AGAGTTAGGCTGCTGCTGGTTGGC-3′ for GmERF3 and sense 5′-CCAAGCAGAAGAAGATGATG-3′and antisense 5′-ACCAGTACTAGAGTTCTCAC-3′ for GmWRKY54.The soybean constitutively expressed CYP2(cyclophilin) gene was used as a reference for normalization.The sequences of the primers were as follows: sense 5′-CGGGACCAGTGTGCTTCTTCA-3′ and antisense 5′-CCCCTCCACTACAAAGGCTCG-3′ (Ni et al.2013).The CYP2 gene was also used to normalize the expression levels of GmNAC15 in the hairy roots.

2.5.Detection of cell death

Programmed cell death was analyzed by Trypan Blue(Huang et al.2011).Leaves from the untreated and salttreated overexpression and control plants were soaked in a 0.4% Trypan Blue solution (MYM Biological Technology Co., Ltd., Beijing, China), boiled for 2 min in a water bath, and then incubated for 8 h.Then, the leaves were transferred to a solution with 1.25 g mL-1chloral hydrate(MYM Biological Technology Co., Ltd.) for fading for three days, and the chloral hydrate solution was changed once every day.

2.6.Hydrogen peroxide and superoxide staining

2.7.Measurement of chlorophyll content

For the determination of chlorophyll content, the leaves of the untreated and salt-treated control and overexpression plants were used.Each leaf sample comprised nine leaves from three different plants.Chlorophyll content was measured using a modified method described by Aono et al.(1993).

2.8.Measurement of malondialdehyde content

The measurement of malondialdehyde (MDA) content was conducted according to the TBA method described by Puckette et al.(2007).Each root sample comprised nine roots from three different plants.All measurements were repeated three times.

2.9.Measurement of betaine content

Samples of 0.5 g roots from the untreated and salt-treated control and overexpression plants were soaked in 10 mL 95% ethanol.The concentrate was heated for 3 h and then diluted with 3 mL 0.1 mol L-1HCl, followed by two extractions with 0.3 and 0.5 mL petroleum ether.Active carbon was added to decolorize the solution.After centrifugation,2 mL supernatant was transferred to a new tube with 4 mL Reinecke’s salt, and the solution was cooled for 1 h at 4°C.After centrifugation, the supernatant was removed, and the precipitate was washed with 99% diethyl ether.The homogenate was centrifuged at 4 000 r min-1for 15 min, and then, the precipitate was dissolved with 1 mL 70% acetone.The absorbance of the solution was read at 525 nm.Each root sample consisted of nine roots from three different plants.All measurements were repeated three times.The betaine content was calculated as follows:

Betaine content=(OD525-0.0121)/0.035×3×10/0.5

3.Results

3.1.Cloning and sequence analysis of GmNAC15

The cDNA sequence of GmNAC15 was 1 412 bp in length,including a complete open reading frame (ORF) of 912 bp with a 5′-untranslated region (UTR) of 121 bp and 3′ UTR of 379 bp, encoding a protein of 303 amino acids with a predicted molecular mass of 34.3 kDa and a predicted pI of 8.51.

A comparison of amino acid sequences of the N-terminal subdomains showed that GmNAC15 proteins are 35.71-44.19% identical to Arabidopsis AtNAC proteins.Subdomains A, C, and D are highly conserved, subdomains B and E are divergent (Appendix A).Phylogenetic analysis indicated that GmNAC15 clustered with AtNAC2 (Appendix B), of which AtNAC2 could enhance salt tolerance of groundnut (Patil et al.2014).

3.2.Expression pattern of GmNAC15 gene in various tissues and in response to various stresses

qRT-PCR was used to analyze the tissue-specific expression of GmNAC15.As shown in Fig.1-A, GmNAC15 was constitutively expressed in the roots, stems, leaves,cotyledons, and flower tissues.The GmNAC15 expression level was higher in roots than in any other tissues, except the cotyledons.

The expression pattern of the GmNAC15 gene was detected in the soybean roots (Fig.1-B) and leaves(Fig.1-C) after exposure to various abiotic stressors.In the root tissue under NaCl stress, GmNAC15 reached peak expression at 6 h, decreased significantly at 12 h,and increased again at 24 h.Under PEG stress, GmNAC15 reached peak expression at 6 h, decreased significantly at 12 h, and increased at 24 h.Under the ABA treatment, the transcription level of GmNAC15 increased rapidly and peaked at 24 h.Under cold stress, the expression level of GmNAC15 decreased gradually and reached the lowest point at 24 h.In the leaf tissues under NaCl stress, the transcription level of GmNAC15 increased gradually, and after 6 h,the expression level of GmNAC15 increased rapidly and peaked at 24 h.Under PEG stress, the expression level of GmNAC15 increased rapidly, peaked at 12 h and then declined.Under ABA treatment, the transcription level of GmNAC15 decreased to the lowest point at 0.5 h and then increased significantly.Under cold stress, the expression level of GmNAC15 increased gradually, peaked at 2 h, and then decreased rapidly, reaching the lowest point at 24 h.These results suggest that the expression of GmNAC15 is responsive to salt, dehydration, cold, and exogenous ABA treatments in both roots and leaves.

Fig.1 Expression patterns of GmNAC15 in various tissues and in response to various stresses.A, the transcription level of GmNAC15 in different soybean tissues was measured by qRT-PCR (R, root; S, stem; L, leaf; C, cotyledons; F, flower).B, expression patterns of GmNAC15 after exposure to various abiotic stressors in soybean roots.C, expression patterns of GmNAC15 after exposure to various abiotic stressors in soybean leaves.PEG, polyethylene glycol; ABA, abscisic acid.Values represent the averages of three independent biological experiments, and error bars represent standard deviations.Asterisks indicate a significant difference(＊, P＜0.05; ＊＊, P＜0.01) compared with the corresponding controls.

3.3.Overexpression of GmNAC15 confers salt tolerance in soybean

To examine the transcription level of GmNAC15 in the VC and OE plants, samples from the hairy roots and leaves of one-mon-old VC and OE plants infected with A.rhizogenes K599 were assessed by qRT-PCR.Every VC and OE sample contained 10 soybean plants.In the hairy roots,qRT-PCR (Fig.2-A) revealed that the expression level of GmNAC15 in the OE plants was upregulated at least 3-fold compared to VC plants.In the leaves, qRT-PCR (Fig.2-B)showed that there was no significant difference between the VC and OE plants.One potential explanation is that overexpression of GmNAC15 in hairy root couldn’t influnce on the expression level of GmNAC15 in leaf.

To analyze the role of GmNAC15 in salt tolerance,45-day-old soybean plants were infected with A.rhizogenes K599 and irrigated with 250 mmol L-1NaCl.After five days,the VC plants displayed severe wilting, the survival rate is 43%; however, the condition of the OE plants was better than that of the VC plants, the survival rate is 73% (Fig.2-C).These results showed that overexpression of GmNAC15 in hairy roots can enhance the salt tolerance of soybean.

3.4.The different physiological indexes between vector control and overexpression plants

Trypan blue staining revealed no obvious differences between the leaves of the VC and OE plants before salt stress; however, VC leaves were stained to a greater extent relative to the OE leaves after two days of 250 mmol L-1NaCl treatment (Fig.3-A).The salt stress potentially resulted in more severe cell death in the VC leaves.The VC and OE leaves were stained with DAB and NBT solution specifically to detect the accumulation ofandThere was almost no brown precipitate and blue spots inthe VC and OE leaves before salt stress; however, the OE leaves accumulated markedly lower levels ofandthan the VC leaves after two days of salt stress (Fig.3-B and C).Taken together, these data suggested that any injury caused by ROS may be effectively alleviated by overexpression of GmNAC15 in hairy roots.The chlorophyll content of VC leaves was similar to that of OE leaves before salt stress.Although the chlorophyll content of both the VC and OE leaves decreased after two days of salt stress,the chlorophyll content of the VC decreased more rapidly(Fig.3-D).This finding suggests that the function of wild leaves is influenced by transgenic hairy roots.

Fig.2 Overexpression of GmNAC15 confers salt tolerance to soybean plants.A, in hairy roots, the expression levels of GmNAC15 in both the vector control (VC) and overexpression composite (OE) plants were detected using qRT-PCR.B, in leaves, the expression of GmNAC15 in both VC and OE plants were detected using qRT-PCR.C, a 45-d-old soybean plant was infected with Agrobacterium rhizogenes K599 and irrigated with 250 mmol L-1 NaCl.The photographs were taken at five days after salt treatment.OE1, OE2,and OE3 represent three repeated experiments.Values represent the averages of three independent biological experiments, and error bars represent standard deviations.＊＊ indicates a significant difference at P＜0.01 compared with the corresponding controls.

The VC and OE roots had the same MDA content before salt stress.Although the MDA content of both the VC and OE hairy roots increased after exposure to salt stress, the MDA content of the OE hairy roots was lower than that of the VC hairy roots (Fig.3-E).There were no significant differences in betaine content between VC hairy roots and OE hairy roots before salt stress.Although the betaine content of both the VC and OE hairy roots was upregulated after two days of salt stress, the betaine content was significantly higher in the OE hairy roots than in the VC hairy roots (Fig.3-F).These data suggest that any injury caused by salt stress may be effectively alleviated by overexpression of GmNAC15 in hairy roots.

3.5.GmNAC15 regulates salt stress-responsive gene expression

GmERF3 is a newly discovered member of the AP2/ERF transcription factor family and plays an important role in salt stress (Zhang et al.2009).GmWRKY54 belongs to the WRKY-type transcription factor family and enhances the salt and drought tolerance of Arabidopsis, possibly through the regulation of DREB2A and STZ/Zat10 (Zhou et al.2008).The expression of GmERF3 and GmWRKY54 was significantly higher in the OE hairy roots than in the VC hairy roots (Fig.4-A and B).These results indicate that GmNAC15 may confer salt tolerance through regulation of salt stress-responsive genes.

4.Discussion

Fig.4 The expression of salt stress-related genes.A, the transcription level of GmERF3.B, the transcription level of GmWRKY54.Total RNA was isolated from the hairy roots of the vector control (VC) and overexpression composite (OE) plants before salt stress.Values represent the averages of three independent biological experiments, and error bars represent standard deviations.Asterisks indicate a significant difference (＊, P＜0.05; ＊＊, P＜0.01) compared with the corresponding controls.

The NAC (NAM, ATAF1/2 and CUC2) family is a large class of TFs that play important regulatory roles in plant development and responses to abiotic stress.Increasing evidence has suggested that the NAC family members can enhance salt tolerance in a number of plants, such as rice (Zheng et al.2009), wheat (Mao et al.2012),and Arabidopsis (He et al.2005); however, studies of soybean NAC genes remain limited.Our data indicated that overexpression of GmNAC15 in hairy roots enhances soybean salt tolerance.

GmNAC15 in hairy roots and leaves is regulated by salt, drought, cold, and exogenous ABA, suggesting that GmNAC15 might be involved in stress responses.Comparing these stress treatments, we found that the transcription level of GmNAC15 was induced in response to 250 mmol L-1NaCl, 20% PEG, and 100 μmol L-1ABA in both roots and leaves (Fig.1-B and C), and especially in leaves after salt stress, the expression level of GmNAC15 gradually increased with time.Since the transformation of soybean plants is extremely difficult and time-consuming, we overexpressed GmNAC15 in hairy roots using A.rhizogenes K599.45-day-old composite soybeans were irrigated with 250 mmol L-1NaCl.The OE plants displayed a higher survival rate than the VC plants (Fig.2-C).In the roots,qRT-PCR (Fig.2-A) showed that the GmNAC15 expression in the OE plants was upregulated at least thrice compared to the VC plants.These findings demonstrate that GmNAC15 is a positive regulator of salt tolerance.

Salt stress can lead to the formation ofandElevations in cytotoxic oxygen will disturb normal metabolism through oxidative damage to the cell membrane structure, which ultimately results in cell death.We detected more brown precipitate (Fig.3-B) and blue spots(Fig.3-C) in the VC leaves than in the OE leaves after salt stress.This finding suggested that the VC leaves suffered more serious ROS damage; thus, the VC leaves experienced more cell death than the OE leaves (Fig.3-A).In addition, the degradation rate of chlorophyll content in the VC leaves was faster than that of the OE leaves after salt stress (Fig.3-D).In the leaves, qRT-PCR (Fig.2-B)showed that the GmNAC15 expression of the OE plants was not significantly different from that of the VC plants.This finding suggested that the function of the wild leaves was influenced by the transgenic hairy roots.Plant cells accumulate MDA because of lipid peroxidation caused by ROS, and thus, MDA is used to estimate ROS-mediated injuries in plants.Cell membrane stability is affected when lipids are damaged by ROS.Cell membrane stability also serves as an indicator of salt tolerance.As shown in Fig.3-E, the lipid peroxidation induced by ROS injury was relatively moderate in the OE hairy roots.We hypothesize that OE hairy roots have an enhanced antioxidant defense system.When suffering from salt stress, many plants can accumulate more compatible osmolytes, including betaine and soluble sugars, which play roles in osmotic adjustment.After 250 mmol L-1NaCl treatment, the OE hairy roots accumulated much more betaine than the VC hairy roots(Fig.3-F).This finding indicated that GmNAC15 is involved in regulating the synthesis of osmolytes to reduce osmotic stress in OE plants.

GmERF3 is induced by salt stress and plays an important role in salt stress.Overexpression of GmWRKY54 enhances Arabidopsis salt tolerance.The expression levels of GmERF3 and GmWRKY54 were elevated in the OE hairy roots (Fig.4-A and B).These results indicated that GmNAC15 may confer tolerance to salt stress through the regulation of salt stress-responsive genes in the OE hairy roots.

5.Conclusion

Our data indicate that overexpression of GmNAC15 in hairy roots enhances salt tolerance.Transgenic hairy roots could influence the function of wild leaves; however, overexpression of GmNAC15 in hairy root couldn’t influnce on the expression level of GmNAC15 in leaf.These results suggest that GmNAC15 likely functions as a positive regulator of salt tolerance.

Appendicesassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

Acknowledgements

This study was supported by the National Key Research and Development Program of China (2016YFD0101005)and the Agricultural Science and Technology Program for Innovation Team on Identification and excavation of Elite Crop Germplasm, Chinese Academy of Agricultural Sciences.

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