The NAC-like transcription factor SiNAC110 in foxtail millet (Setaria italica L.) confers tolerance to drought and high salt stress through an ABA independent signaling pathway

Journal of Integrative Agriculture 2017, 16(3): 559–571 Available online at www.sciencedirect.com

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RESEARCH ARTICLE

The NAC-like transcription factor SiNAC110 in foxtail millet (Setaria italica L.) confers tolerance to drought and high salt stress through an ABA independent signaling pathway XIE Li-na1*, CHEN Ming2*, MIN Dong-hong1, FENG Lu1, XU Zhao-shi2, ZHOU Yong-bin2, XU Dong-bei1, LI Lian-cheng2, MA You-zhi2 , ZHANG Xiao-hong3 1

College of Agronomy, Northwest A&F University, Yangling 712100, P.R.China National Key Facility for Crop Gene Resources and Genetic Improvement, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China 3 College of Sciences, Northwest A&F University, Yangling 712100, P.R.China 2

Abstract Foxtail millet (Setaria italica (L.) P. Beauv) is a naturally stress tolerant crop. Compared to other gramineous crops, it has relatively stronger drought and lower nutrition stress tolerance traits. To date, the scope of functional genomics research in foxtail millet (S. italic L.) has been quite limited. NAC (NAM, ATAF1/2 and CUC2)-like transcription factors are known to be involved in various biological processes, including abiotic stress responses. In our previous foxtail millet (S. italic L.) RNA seq analysis, we found that the expression of a NAC-like transcription factor, SiNAC110, could be induced by drought stress; additionally, other references have reported that SiNAC110 expression could be induced by abiotic stress. So, we here selected SiNAC110 for further characterization and functional analysis. First, the predicted SiNAC110 protein encoded indicated SiNAC110 has a conserved NAM (no apical meristem) domain between the 11–139 amino acid positions. Phylogenetic analysis then indicated that SiNAC110 belongs to subfamily III of the NAC gene family. Subcellular localization analysis revealed that the SiNAC110-GFP fusion protein was localized to the nucleus in Arabidopsis protoplasts. Gene expression profiling analysis indicated that expression of SiNAC110 was induced by dehydration, high salinity and other abiotic stresses. Gene functional analysis using SiNAC110 overexpressed Arabidopsis plants indicated that, under drought and high salt stress conditions, the seed germination rate, root length, root surface area, fresh weight, and dry weight of the SiNAC110 overexpressed lines were significantly higher than the wild type (WT), suggesting that the SiNAC110 overexpressed lines had enhanced tolerance to drought and high salt stresses. However, overexpression of SiNAC110 did not affect the sensitivity of SiNAC110 overexpressed lines to abscisic acid (ABA) treatment. Expression analysis of genes involved in proline synthesis, Na+/K+ transport, drought responses, and aqueous transport proteins were higher in the SiNAC110 overexpressed lines than in the WT, whereas expression of ABA-dependent pathway genes did not change. These results

Received 5 April, 2016 Accepted 20 May, 2016 XIE Li-na, Mobile: +86-15801247289, E-mail: xlna163@163. com; CHEN Ming, Tel: +86-10-82108750, E-mail: chenming02@ caas.cn; Correspondence ZHANG Xiao-hong, Mobile: +8613572869993, E-mail: [email protected]; MA You-zhi, Tel: +86-10-82108750, E-mail: [email protected] * These authors contributed equally to this study. © 2017, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(16)61429-6

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indicated that overexpression of SiNAC110 conferred tolerance to drought and high salt stresses, likely through influencing the regulation of proline biosynthesis, ion homeostasis and osmotic balance. Therefore, SiNAC110 appears to function in the ABA-independent abiotic stress response pathway in plants. Keywords: foxtail millet (Setaria italica (L.), NAC-like transcription factor, drought stress, high salt stress, ABA-independent pathway

1. Introduction Soil salinity and drought stress have a strongly negative influence on plant growth and crop yield (Fry 2008; Hwang et al. 2012; Suzuki et al. 2016). Plant responses to abiotic stresses are known to be regulated by proteins of many transcription factor families, including the AP2/ERF, bZIP, NAC, MYB, MYC, Cys2His2, zinc-finger, and WRKY families (Yamaguchi-Shinozaki and Shinozaki 2006; Udvardi et al. 2007; Nakashima et al. 2009; Xu et al. 2011). The NAC (NAM, AFAT 1/2, andCUC2) transcription factors are in a plant-specific protein family. These proteins were known to function in important roles in various biological processes, including growth and development, cell division, senescence, and both biotic and abiotic stress responses in plants (Souer et al. 1996; Aida et al. 1997; Xie et al. 1999, 2000; Ren et al. 2000; Hegedus et al. 2003; Olsen et al. 2005). The NAC-like transcription factors have a characteristic NAM domain at their N-terminal regions and different transcription regulation domains at their C-terminal regions. NAC-like transcription factor genes, including ONAC022, SNAC1 and ANAC036, are also known to be involved in stress responses in plants, and their transcriptional activities have been shown to depend on their respective C-terminal regions (Hu et al. 2006; Kato et al. 2010; Hong et al. 2016). Abscisic acid (ABA) is an important phytohormone involved in the regulation of abiotic stress responses in plants. Two ABA signaling pathways mainly control abiotic stress responses: the ABA dependent pathway and the ABA independent pathway (Yamaguchi-Shinozaki and Shinozaki 2006). bZIP-like transcription factors are often involved in the ABA dependent pathway, while DREB-like transcription factors are often involved in regulating the ABA independent pathway (Zhu et al. 2002; Fujita et al. 2005). To date, most of the research about NAC-like transcription factors have focused on gene function. However, the precise roles of NAC-like transcription factors in regulating the ABA-dependent and/ or the ABA-independent pathway remain as yet unknown. Foxtail millet (Setaria italic L.) was domesticated from the weed S. viridis (Zhang et al. 2012). Foxtail millet (S. italic L.) is an excellent biological material with which to study plant mechanisms of tolerance to abiotic stress in gramineous

crops because, compared with other gramineous crops, foxtail millet (S. italic L.) has many physiological characteristics that enable it to thrive in harsh environmental conditions, including strong tolerance to various abiotic stress. Compared with switchgrass, napier grass and pearl millet, foxtail millet (S. italic L.) also has a comparatively simple genome, and its genome sequence is now available to researchers (Bennetzen et al. 2012; Puranik et al. 2013). However, to date, functional genomic research of foxtail millet (S. italic L.) remains quite preliminary. Increased focus on functional genomics research in foxtail millet (S. italic L.) will help to identify candidate genes that can be used for the improvement of tolerance to abiotic stress in gramineous crops (Li and Brutnell 2011; Bennetzen et al. 2012). Puranik et al. (2013) analyzed the expression profiles of 50 foxtail millet (S. italic L.) NAC genes in response to various abiotic stresses, and found that SiNAC110 was up-regulated following both dehydration and salinity stress; its expression level increased more than 9-fold in stress-treated plants. In this study, we also found that stress treatment induced the expression of the SiNAC110 transcription factor in foxtail millet (S. italic L.). Overexpression of SiNAC110 enhanced tolerance to both high salt stress and drought stress in transgenic Arabidopsis. However, of note, SiNAC110 overexpression lines were not sensitive to ABA, and the expression of genes related to the ABA dependent pathway did not differ between SiNAC110 overexpression lines and WT plants, suggesting that SiNAC110 overexpression lines conferred tolerance to drought and high salt stress through the ABA independent pathway. These results represent new knowledge about the regulatory mechanisms of abiotic stresses response in foxtail millet (S. italic L.) and suggest that SiNAC110 may be a powerful genetic resource for use in abiotic stress resistance breeding programs in gramineous crops.

2. Materials and methods 2.1. Phylogenetic tree and protein domain analysis According to the results of foxtail millet (S. italic L.) RNA seq analysis, we found the drought up-regulated gene SiNAC110 (Si026530m.g) and then obtained the gene sequence from the Phytozome web site (http://phytozome.jgi.doe.gov/pz/

XIE Li-na et al. Journal of Integrative Agriculture 2017, 16(3): 559–571

portal.html). The sequences of SiNAC110 homologous genes from Oryza sativa (Os), Triticum aestivum (Ta), Zea mays (Zm), and S. italica (Si), were all downloaded from the Phytozome web site (http://phytozome.jgi.doe.gov/pz/portal. html) and the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). The SiNAC110 protein sequence was downloaded from NCBI (http://www.ncbi.nlm. nih.gov/). Multiple sequence alignment of the full-length amino acid sequences of the SiNAC110 proteins were performed using Clustal X, and phylogenetic trees were generated using MEGA5.0. The online SMART program (http:// smart.embl-heidelberg.de/) was used to analyze the SiNAC protein domains. The 5´ flanking regions (approximately 2 000 bp) of the SiNAC110 gene was downloaded from the Phytozome web site. The putative cis-acting regulatory elements of SiNAC110 were identified using the PLACE database (http://www.dna.affrc.go.jp/PLACE/).

2.2. Quantitative RT-PCR and subcellular localization analysis Real-time quantitative PCR analysis was used to measure the SiNAC110 expression level in different treatment groups. 10-d-old foxtail millet (S. italic L.) seedlings were cultured on Murashige and Skoog (MS) medium, to which the following treatments were amended: 20% PEG6000, 200 mmol L–1 NaCl, 100 μmol L–1 H2O2, 100 μmol L–1 ABA, 100 μmol L–1 salicylic acid (SA), and 0.3 mmol L–1 N (low nitrogen). A low temperature treatment was also performed (4°C). The samples were harvested at 0, 1, 6, 12, 24, and 48 h, and RNA was isolated from the plants using a Plant RNA Kit (Tiangen, China). Total RNA was used as the template to synthesize cDNA using TransScript First-Strand cDNA Synthesis SuperMix (Transgene Biotech, China). Real-time quantitative PCR was performed using an ABI 7500 Real-Time PCR System. Foxtail millet (S. italic L.) SiActin (Si001873m.g) was used as an internal control. The primer pairs used in the real-time quantitative PCR analysis are listed in Appendix A. Three independent biological replicates were analyzed. In the subcellular location assays, which used the protoplast preparation protocol reported by method of Yoo et al. (2007), SiNAC110 was inserted into the p16318 vector and thereby fused with the sequence encoding green fluorescent protein (GFP) to yield SiNAC110-GFP. The empty p16318: GFP vector was used as a control. The protoplasts were observed using a confocal laser-scanning microscope (ZEISS LSM 700; Germany).

2.3. Cloning of SiNAC110 and overexpression of SiNAC110 in Arabidopsis The total RNA was extracted and used to synthesize

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cDNA, as described above. The primers F: 5´-GAGGATC CCCGGGATGCCGATCGCCAGC-3´ and R: 5´-ACTAGTG GATCCCCCGGGCTACTGCATCTCGATG-3´ were used to amplify the SiNAC110 gene. SiNAC110 was inserted into the pBI121vector (driven by the CaMV 35S promoter). The Agrobacterium-mediated floral dipping method (Clough and Bent 1998; Zhong et al. 2015) was used to transform wild type (Col-0) Arabidopsis. T3 generation transgenic plants were used for the stress experiments.

2.4. Phenotype analyses for drought and salt tolerance and ABA sensitivity For the seed germination assays, surface-sterilized wild type (Col-0, WT) and overexpression (OE) seeds were plated on the following media: modified MS medium (containing 3% sucrose and 0.45% phytagel), MS plus 4% PEG6000, MS plus 6% PEG6000, as well as MS medium with two different concentrations of NaCl (100 and 150 mmol L–1), and MS medium supplemented with two different concentrations of ABA (0.6 and 1.0 µmol L–1). After stratification for 3 d at 4°C in the dark, the plates were incubated at 22°C with a 16/8 h light/dark photoperiod. Phenotypes were observed, and data for germination rate determination were recorded every 12 h, over a total period of 96 h. For the phenotypic analysis of seedlings, WT and OE seeds were grown on MS medium for 7 d, and then treated with MS plus 4% PEG6000, 8% PEG6000, two concentrations of NaCl (100 and 130 mmol L–1), and MS medium supplemented with 10 µmol L–1 ABA, with MS medium as a control. After 12 d of treatment, we measured root length, root surface area, fresh weight, and dry weight.

2.5. Expression analysis of selected stress- and ABA-related genes Seeds of WT and OE plants were grown to the 4-leaf stage and then treated in MS medium plus 4% PEG and 100 mmol L–1 NaCl for 24 h. Total RNA was isolated as described above. Quantitative real-time PCR was used to analyze the expression level of selected stress- and ABA-related genes.

3. Results 3.1. SiNAC110 is a stress-responsive NAC gene in foxtail millet (S. italic L.) In foxtail millet (S. italic L.), the 147 members comprising the NAC gene family are distributed onto the nine foxtail millet (S. italic L.) chromosomes. SiNAC110 is located on chromosome 8 (Puranik et al. 2013). The expression of

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SiNAC110 (Si026530m.g) is known to be up-regulated by

elements (W-BOXWRK), 4 drought responsive elements

drought stress conditions. SiNAC110 is 1 101 bp in length

(MYB), 3 ABA and cold responsive elements (MYC), 17

and is predicted to encode a protein of 366 amino acids

low temperature responsive elements (LTRE), and 5 salt

with a molecular weight of 40.808 kD and an isoelectric

stress responsive elements (GT) in the promoter region

point of 6.31. SiNAC110 has a conserved NAM domain

of SiNAC110 (Appendix B).

between amino acids 11–139 (Fig. 1). SiNAC110 has three exons and two introns. Among the genes of the foxtail millet (S. italic L.) NAC gene family, SiNAC110

3.2. Expression pattern and subcellular localization analysis of SiNAC110

shares the highest homology SiNAC105. Phylogenetic analysis indicated that the NAC gene family is divided

Our qRT-PCR analysis indicated that the transcription of

into five subgroups, from I to V; SiNAC110 belongs to

SiNAC110 in plants treated with drought stress gradually

subgroup III (Fig. 2). SiNAC110 promoter analysis was

increased, peaking at 24 h. The expression of SiNAC110

completed using PLACE online tools (http://www.dna.affrc.

was 28-fold higher in the drought stress treated plants

go.jp/PLACE/). This analysis showed that there are 11

than in the untreated control plants (Fig. 3-C). Under high

ABA-responsive elements (ABRE), 18 wound responsive

salt treatment, the expression of SiNAC110 increased and

A SiNAC110 SiNAC105

NAM ***********************************************************:******************** MPIASSRLPNLPAGFRFHPTDEELIVHYLMNQASSLPCPVPIIAEVNIYQCNPWDLPAKSLFGENEWYFFSPRDRKYPNG MPIASSRLPNLPAGFRFHPTDEELIVHYLMNQASSLPCPVPIIAEVNIYQCNPWDLPAKALFGENEWYFFSPRDRKYPNG 1.......10........20........30........40........50........60........70........80

80 80

NAM SiNAC110 SiNAC105

*************************************************************** **************** ARPNRAAGSGYWKATGTDKAILSTPTSENIGVKKALVFYGGKPPKGTKTDWIMHEYRLTGANKGTKRRGSSMRLDDWVLC 160 ARPNRAAGSGYWKATGTDKAILSTPTSENIGVKKALVFYGGKPPKGTKTDWIMHEYRLTGANKTTKRRGSSMRLDDWVLC 160 ........90.......100.......110.......120.......130.......140.......150.......160

SiNAC110 SiNAC105

************* ************************ ******* ********************************* RIYKKSNNFQFSDQDQEGSTVEEESLNNNMNSTSAASPNKSDANDH-DDQFQFQPTTMSMSKSYSITDLLNTIDYSALSQ 239 RIYKKSNNFQFSDPDQEGSTVEEESLNNNMNSTSAASP-KSDANDHNDDQFQFQPTTMSMSKSYSITDLLNTIDYSALSQ 239 .......170.......180.......190.......200.......210.......220.......230.......240

SiNAC110 SiNAC105

****** *********************************:************************ ************** LLDAPAEAEPPLIYPTTTQTHQSLNYNNNVMNNNSHFNLPEAADACPDYVAPNNCNGLKRKRVMTMDGAESSFDDGSRKL 319 LLDAPAAAEPPLIYPTTTQTHQSLNYNNNVMNNNSHFNLPQAADACPDYVAPNNCNGLKRKRVMTTDGAESSFDDGSRKL 319 .......250.......260.......270.......280.......290.......300.......310.......320

SiNAC110 SiNAC105

** **********.********************* ************ LK-LPSDSRSSGHGHFVGSTSSYCNQQLVDTSGFQYSSLLSYPFIEMQ LKLLPSDSRSSGHSHFVGSTSSYCNQQLVDTSGFQCSSLLSYPFIEMQ .......330.......340.......350.......360........

B

366 367

SiNAC067 SiNAC084 SiNAC031 SiNAC055 SiNAC110 SiNAC105 SiNAC012 SiNAC013

Fig. 1 Sequence alignment and protein domain analysis of foxtail mllet (Setaria italic L.) SiNAC110 with related proteins. A, aligned sequences of SiNAC110 and SiNAC105, the horizontal line indicates a conserved NAM (no apical meristem) domain at the 11–139 amino acid position. B, protein domain analysis of SiNAC110 with related foxtail millet (S. italic L.) proteins. Blue indicates transmembrane domains, gray indicates NAM domains.

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2G 100

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TaNAC29 ONAC010 ZmNAC100

Fig. 2 Phylogenetic analysis of NAC-like proteins from S. italica (Si), Oryza sativa (Os), Triticum aestivum (Ta), and Zea mays (Zm). The NAC protein sequences of these plants were downloaded from the Phytozome ver. 10.0.4 database.

peaked at 48 h (70-fold higher expression than the control) (Fig. 3-D). Under the low nitrogen and cold stress treatments, the expression of SiNAC110 increased by 7-fold

3.3. Overexpression of SiNAC110 improved tolerance to drought and salt stresses in transgenic Arabidopsis

(Fig. 3-A) and 13-fold, respectively, compared to the controls (Fig. 3-G). SiNAC110 expression was also induced by

The results of tolerance to drought stresses analysis

H2O2, ABA and salicylic acid (SA) treatments (Fig. 3-E and

showed that, on normal MS medium, the seed germination

F). Our subcellular localization analysis showed that the

of SiNAC110 transgenic plants and WT germinated nor-

SiNAC110-GFP fusion protein was localized to the nucleus;

mally (Fig. 4-A and D). On the medium plus 4% PEG6000

the GFP control was present in the membrane, cytoplasm,

and 6% PEG6000, the germination rate of the transgenic

and nucleus (Fig. 3-H).

plants was higher than that of the WT seeds (Fig. 4-B, C,

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Fig. 3 Expression patterns of SiNAC110 and subcellular localization of the SiNAC110-GFP fusion protein. A–G, expression patterns of SiNAC110 at 0, 1, 6, 12, 24, and 48 h with different treatments. A, low nitrogen stress (0.3 mmol L–1 nitrogen). B, 20% PEG6000. C, 100 mmol L–1 NaCl. D, low temperature (4°C). E,100 mmol L–1 hydrogen peroxide. F, 100 mmol L–1 abscisic acid (ABA). G, 100 mmol L–1 salicylic acid (SA). Values are means±standard deviation (SD) (n=3 independent experiments). Bars superscripted by different capital letters represent significant differences at the P<0.01 level. The same as below. H, visualization with confocal microscopy of 35S: SiNAC110-GFP and 35S: GFP control vectors transiently transformed into Arabidopsis protoplasts cells.

E, and F). During the seedling growth stage, SiNAC110

SiNAC110 transgenic and WT plants germinated normally

transgenic plants and WT grew normally on MS medium.

on MS medium (Fig. 5-A and D), whereas on MS plus 100

On the MS plus 4 and 8% PEG6000 media, the transgen-

and 150 mmol L–1 NaCl medium, the germination of WT

ic plants grew better than WT (Fig. 4-G–I), and the root

seeds was significantly inhibited as compared to transgenic

lengths, root surface areas, fresh weights, and dry weights

seeds (Fig. 5-B, C, E, and F). During the seedling stage, the

of the transgenic plants were significantly larger than those

transgenic plants grew better than WT on MS plus 100 mmol

of the WT plants (P<0.05) (Fig. 4-J–M). These results in-

L–1 NaCl, and 130 mmol L–1 NaCl medium, (Fig. 5-H and I),

dicated that SiNAC110 conferred drought tolerance during

whereas seedlings of SiNAC110 transgenic and WT plants

germination and the seedling stage in transgenic plants.

grew normally; no significant differences were observed

For the salt stress tolerance analysis, the seed of

on MS medium (Fig. 5-G). The root length, root surface

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Fig. 4 Germination, seeding root length, root surface area, fresh weight, dry weight of wild type (WT) and overexpressing (OE) under drought stress. A–C, OE and WT plants grown on Murashige and Skoog (MS) medium, MS plus 4% PEG6000 and MS plus 6% PEG6000, respectively. D–F, seed germination rates under MS, MS plus 4% PEG6000, and MS plus 6% PEG6000 medium, respectively. G–I, seeding growth of OE and WT plants on MS, MS plus 4% PEG6000 and MS plus 8% PEG6000 medium, respectively. Statistical analysis of data for OE and WT are shown for root length (J), root surface area (K), fresh weight (L), and dry weight (M). Bars superscripted by different lowercase letters represent significant differences at the P<0.05 level. The same as below.

area, fresh weight, and dry weight of the transgenic plants

3.4. High-level expression of stress related genes

were all significantly larger than those of the WT (P<0.05)

contributes tolerance to drought and salt stress in

(Fig. 5-J–M). These results indicate that the overexpres-

the SiNAC110 transgenic plants

sion of SiNAC110 conferred tolerance to high salt stress in transgenic plants.

Our qRT-PCR analysis revealed that, under high salt

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WT

OE1

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Fig. 5 Germination, seeding root length, root surface area, fresh weight, and dry weight of WT and OE plants grown under salt stress. A–C, OE and WT plants grown on MS, MS plus 100 mmol L–1 NaCl and 150 mmol L–1 NaCl medium. D–F, seed germination rates under the MS, MS plus 100 mmol L–1 NaCl and 150 mmol L–1 NaCl medium. G–I, seeding growth of OE and WT plants on MS, MS plus 100 mmol L–1 NaCl and 130 mmol L–1 NaCl medium.

treatment, the expression of some ion transporter genes, including HKT1 (high-affinity K+ transporter) and NHX1 (Na+/ H+ exchanger), was higher (6–10 folds) in the transgenic

plants than in the WT plants (Fig. 6-A and C). SOS1 is essential for the homeostasis of both Na+ and K+ (Ding and Zhu 1997). The expression of SOS1 increased in SiNAC110

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transgenic plants in high salt treatment compared to WT plants (Fig. 6-B). Under drought treatment, the expression of some key regulators of proline biosynthesis, including P5CR (delta 1-pyrroline-5-carboxylate reductase) and P5CS2 (delta 1-pyrroline-5-carboxylate synthetase B), was higher (2–3 folds) in transgenic plants than in the WT plants (Fig. 6-F–H). The expression of the important drought-related transcription regulator CBF3 (Dubouzet et al. 2003; Oraby and Ahmad 2012) was 5-fold higher in the transgenic plants than in the WT plants (Fig. 6-E). The expression of the water channel protein gene PIP2A (plasma membrane intrinsic protein 2) was 2.3-fold higher in SiNAC110 transgenic plants than in the WT plants (Fig. 6-D).

4 2

OE2

b

a

b

WT

4

OE1 AtP5CR a

H a

2 b

OE1

OE2

8

OE1

F

a a

4 2

b WT

1.5

1.0

OE1

I

a

a

0.5

0.0

2

OE1

OE2

a

OE1

OE2

b

WT

2.0

AtP5CS2 a

a

1.5 1.0

b

0.5

WT

1.5

1.0

OE1

OE2

AtABI5 a

a

a

OE1

OE2

0.5

0.0 WT

AtNHX1 a

4

0.0

OE2

AtABI4 a

6

0

OE2

AtCBF3

6

0

OE2

3

WT

WT E

AtPIP2A

2

0

a

6

Relative expression level

OE1

a

1

8

C

0 WT

3

1

a

AtHKT1

10

Relative expression level

Relative expression level Relative expression level

b

Relative expression level

2

Phenotype analysis indicated that there were no significant differences among several traits between the SiNAC110 transgenic plants and WT plants grown on MS medium or on MS medium supplemented with 0.6, 1.0 or 10 µmol L–1 ABA (Fig. 7); the traits examined were germination rate (Fig. 7-A–C), root length (Fig. 7-F), and root surface area (Fig. 7-G). There were no significant differences between SiNAC110 transgenic and WT plants under ABA treatment. We also found that, under drought and salt stress, the expression of some ABA-dependent pathway related genes

Relative expression level

4

0

G

a

Relative expression level

a

0

D

B

AtSOS1

6

Relative expression level

Relative expression level

A

3.5. SiNAC110 transgenic lines were insensitive to ABA treatment

WT

Fig. 6 Expression analyses of stress-related genes in WT and OE under drought and salt stress. A–C, expression of salt stress related genes, including AtSOS1, AtHKT1 and AtNHX1. D and E, expression of drought stress related genes, including AtCBF3 and AtPIP2A. F and G, expression of proline biosynthesis related genes including AtP5CR and AtP5CS2. H and I, expression of ABA-signaling pathway genes, including AtABI5 and AtABI4.

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Na+ and K+ co-transporter, aids in low-affinity Na+ uptake and high-affinity K+ transport under salt stress conditions (Rubio et al. 1995). NHX1, as a tonoplast Na+/H+ antiporter, can compartmentalize Na+ into the vacuole and thereby alleviate ion toxicity (Zwirn et al. 1997). SOS1 is essential for the homeostasis of both Na+ and K+, and overexpression of SOS1 leads to export of Na+ from the cytosol to the extracellular space and prevents a rapid accumulation of Na+ in the cytoplasm (Ding and Zhu 1997). Our results suggested that SiNAC110 enhanced transgenic plant tolerance to high salt

40 MS+1.0 μmol L–1 ABA

20 0

24

0

60

96

20

80

84

Germination frequency (%)

MS+0.6 μmol L ABA –1

100

12

96

84

48

12

60 72

0

40

96

MS

60

72 84

40

80

36 48

60

C

12 24

80

20

100 Germination frequency (%)

B

60

100

24 36

Germination frequency (%)

A

OE2

OE1

WT

72

Salt and drought stresses cause various physiological changes in plant cells, including alterations of osmotic potential, ion imhomeostasis, cell dehydration, Na+ accumulation, and K+ shortage; these alterations lead to plant cell damage (Apse et al. 1999; Ma et al. 2014). HKT1, as a

60

4. Discussion

36 48

including ABI4 and ABI5 in transgenic lines were not different between the transgenic and WT plants (Fig. 6-G and H).

Time (h) D

WT

OE1

OE2

WT

OE1

OE2

Root lengh (cm)

F 6

Root surface area (cm2)

E

G

a

a

2

0

MS

a

4

MS

MS+10 μmol L–1 ABA

4 3 a

2

a

a

1 0

MS

MS+10 μmol L–1 ABA

MS+10 μmol L–1 ABA

Fig. 7 Phenotype analysis of 35S: SiNAC110 transgenic lines under ABA treatment. A–C, seed germination rates under MS, MS plus 0.6 and 1 µmol L–1 ABA treatments. D and E, OE and WT plants grown on MS medium (D), MS plus 10 µmol L–1 ABA (E). Statistical analysis of data for OE and WT are shown for root length (F) and root surface area (G).

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stress by influencing the Na+/K+ in plant cells. CBF3 plays an important role in drought stress responses; it binds to the dehydration-responsive element (DRE/CRT) to regulate the expression of downstream drought-related genes (Dubouzet et al. 2003; Oraby and Ahmad 2012). AtPIP2A, an aquaporin, facilitates the transport of water across cellular membranes and contributes to the maintenance of the moisture status of plants (Johansson et al. 2000). Abundant accumulation of proline in plant cells can effectively alleviate plant tissue damage that can occur during drought and salt stress conditions (Sakuma et al. 2006). P5CR and P5CS2 are two key enzymes of the proline biosynthesis pathways (Roxas et al. 2000; Lehmann et al. 2010). These results indicated that SiNAC110 enhanced transgenic lines tolerance to drought and high salt stress, likely by altering the expression of genes related to the regulation of osmotic homeostasis and proline biosynthesis. Abscisic acid plays a critical role in regulating abiotic stress responses in plants. In our study, we found that SiNAC110 conferred tolerance to drought and salt stresses in transgenic plants through the ABA independent signaling pathway. Two lines of evidence support this supposition. First, Phenotypic analysis showed SiNAC110 transgenic plants were not sensitive to ABA. Second, there were no differences between the transgenic lines and WT plants in the expression levels of two ABA dependent pathway related genes, AtABI5 and AtABI4, which are known to be positive regulators of abscisic acid signaling (Shu et al. 2013; Cheng et al. 2014). These findings are consistent with several other NAC-like transcription factors. For example, Puranik et al. (2013) found that the expression of SiNAC075, SiNAC065, SiNAC063, and SiNAC0108 in foxtail millet (S. italic L.) was induced by drought and high salt stress treatments, but was not induced by abscisic acid treatment. However, some NAC-like transcription factors also have been found to be involved in the ABA-dependent pathway; examples include ONAC058, ONAC002, and ONAC022 from rice (Hu et al. 2006; Chen et al. 2014; Hong et al. 2016). Overexpression of these three NAC-like transcription factor genes in rice improved the tolerance to abiotic stress, and, of note, the transgenic lines were hypersensitive to exogenously applied ABA. In addition, overexpression of TaNAC29 in Arabidopsis led to enhanced salt and drought tolerance and hypersensitivity to ABA (Huang et al. 2015). These results and our results of the present study, suggest that NAC-like transcription factors can regulate abiotic stress responses through both the ABA-independent and -dependent pathways in plants.

5. Conclusion In this study, we identified a NAC-like transcription factor

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gene, SiNAC110, from foxtail millet (S. italic L.). Subcellular localization analysis revealed that a SiNAC110-GFP fusion protein was localized to the nucleus. The expression of the NAC-like transcription factor gene SiNAC110 was induced by drought, high salinity, and other abiotic stresses in foxtail millet (S. italic L.). Overexpression of SiNAC110 enhanced tolerance to drought and high salt stresses in transgenic Arabidopsis. SiNAC110 likely regulates plant tolerance to drought and high salt stress through regulation of the expression of genes involved in proline biosynthesis, ion homeostasis, and osmotic balance with ABA independent signaling pathway.

Acknowledgements This work was funded by the National Key Project for Research on Transgenic Biology, China (2016ZX08002-002) and the Innovation Project of Chinese Academy of Agricultural Sciences. Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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