TsNAC1 is a Key Transcription Factor in Abiotic Stress Resistance and Growth

36 Pages·2017·1.87 MB·English

Plant Physiology Preview. Published on November 9, 2017, as DOI:10.1104/pp.17.01089
1 Running title: Functional analysis of TsNAC1 in T. halophila
2 Corresponding author details: Juren Zhang; The Key Laboratory of the Plant Cell Engineering
3 and Germplasm Innovation, School of Life Science, Shandong University, Jinan 250100,
4 Shandong, China; Tel: [phone]; Fax: [phone].
5 Title: TsNAC1 is a Key Transcription Factor in Abiotic Stress Resistance and Growth
6 Can Liu1, Baomei Wang1, Zhaoxia Li1, Zhenghua Peng1 and Juren Zhang1, *
7 1The Key Laboratory of the Plant Cell Engineering and Germplasm Innovation, School of Life
8 Sciences, Shandong University, Jinan 250100, Shandong, China
9 *To whom correspondence should be addressed.
10 One-sentence summary: TsNAC1 improves abiotic stress resistance of Thelungiella halophyla by
11 regulating ion transport and retards vegetative growth by limiting the expansion of cells.
12
13 Author contributions: C.L. performed most of the experiments and analyzed the data; B.W. and
14 Z.L. provided technical assistance; Z.P. and J.Z. supervised the experiments and analyzed the data;
15 C.L. and J.Z. completed the writing.
16 Funding information: This work was supported by the National Natural Science Foundation of
17 China [grant no. 31571674 to J.Z.] and National Major Projects for Genetically Modified
18 Organisms Breeding in China [grant no. 2016ZX08003004-003 to J.Z.].
19 Corresponding author email: [email protected]
20
21 Abstract
22 NAC proteins constitute one of the largest families of plant-specific transcription factors, and a
23 number of these proteins participate in the regulation of plant development and responses to
24 abiotic stress. T. HALOPHILA STRESS RELATED NAC1 (TsNAC1) cloned from the halophyte
25 Thellungiella halophila is a NAC transcription factor gene, and its overexpression can improve
26 abiotic stress resistance, especially in the salt stress tolerance, in both T. halophila and
27 Arabidopsis and retard the growth of these plants. In this study, the transcriptional activation
28 activity of TsNAC1 and RD26 from Arabidopsis was compared with the target genes’ promoter
29 regions of TsNAC1 from T. halophila, and the results showed that the transcriptional activation
30 activity of TsNAC1 was higher in tobacco and yeast. The target sequence of the promoter from the
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31 target genes was also identified, and TsNAC1 was shown to target the positive regulators of ion
32 transportation, such as T. HALOPHILA H+-PPASE 1 (TsVP1), and the transcription factors MYB
33 HYPOCOTYL ELONGATION-RELATED (MYBH), HOMEOBOX 12 (HB12). In addition,
34 TsNAC1 negatively regulates the expansion of cells, inhibits LIGHT-DEPENDENT SHORT
35 HYPOCOTYLS 1 (LSH1) and UDP-XYLOSYLTRANSFERASE 2 (TXT2), and directly controls the
36 expression of MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4). Based on these results, we
37 propose that TsNAC1 functions as an important upstream regulator of plant abiotic stress
38 responses and vegetative growth.
39
40 Introduction
41 NAC transcription factors include at least 107 members in Arabidopsis (Riechmann et al., 2000)
42 and constitute one of the largest protein families in higher plants. NAC transcription factors have
43 important roles in plant development (Guo et al., 2005; Petricka et al., 2012), cell apoptosis (Lee
44 et al., 2014), abiotic stress tolerance (Tran et al., 2010), secondary cell wall (SCW) formation
45 (Yamaguchi and Demura, 2010) and endoplasmic reticulum stress resistance (Chi et al., 2017).
46 NAC families have a conserved N-terminal with a similar DNA-binding domain but highly
47 diverse C-terminals, which do not contain any known protein domains (Ooka et al., 2003).
48 Arabidopsis RESPONSIVE TO DESICCATION 26 (RD26, AT4G27410) is a NAC transcription
49 factor belonging to the ATAF subfamily, and it is markedly up-regulated by salt, drought and
50 abscisic acid (ABA) treatment. In addition, RD26 has the ability to positively regulate ABA
51 signaling (Fujita et al., 2004; Tran et al., 2004). In cotton, the overexpression of the rice NAC
52 gene SNAC1 improved tolerance to drought and salt via enhanced root development and reduced
53 transpiration rates ((Liu et al., 2014). Recent studies also showed that in maize, STRESS
54 RELATED NAC1 (ZmSNAC1) worked as a stress-responsive transcription factor in positively
55 modulating abiotic stress tolerance (Lu et al., 2012; Shiriga et al., 2014). However, the response of
56 the downstream networks of these NAC transcription factors (TFs) to abiotic stress have not been
57 clarified.
58 Arabidopsis is a salt-sensitive plant and presents certain limitations in studies of salt and
59 drought tolerance mechanisms. Thellungiella halophila (and Thellungiella salsuginea) is a close
60 relative of Arabidopsis, with similar coding sequences. However, T. halophila does show
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61 differences at the metabolome level, including in the metabolism of alanine and the expression
62 patterns of certain drought or cold responsive genes (Amtmann et al., 2005; Benina et al., 2013). T.
63 halophila is a valuable model plant for the study of plant stress response mechanisms because it
64 has a small genome and exhibits high tolerance to drought, low temperatures and high salinity
65 (Zhu, 2001; Inan et al., 2004).
66 VACUOLAR H+-PYROPHOSPHATASE (H+-PPase) plays an important role in the abiotic stress
67 tolerance of plants. Overexpression of V-PPASE 1 (AVP1; AT1G15690) in Arabidopsis improved
68 the drought and salt tolerance in this species (Gaxiola et al., 2001). The overexpression of T.
69 HALOPHILA H+-PPASE 1 (TsVP1) also enhanced the salt and drought stress resistance in
70 different species, such as cotton, maize and tobacco (Gao et al., 2006; Li et al., 2008; Lv et al.,
71 2009). Although expression of TsVP1 is apparently induced by salt stress, this is not the case for
72 AVP1 in Arabidopsis. Furthermore, one 130 bp (-667 to -538) region was identified as the key
73 sequence for salt stress response from the promoter of TsVP1 in T halophila (Sun et al., 2010).
74 Using the above-mentioned 130 bp region as a starting point, we identified T. HALOPHILA
75 STRESS RELATED NAC1 (TsNAC1), which is an ortholog of RD26 in Arabidopsis. The
76 overexpression of TsNAC1 substantially promoted the abiotic stress tolerance of plants and
77 retarded the growth of Thellungiella plants. We compared differences between TsNAC1 and RD26
78 to determine the function of TsNAC1 in growth regulation and abiotic responses. A ChIP-Seq
79 assay was used to reveal the downstream genes and regulation network. This study provides
80 evidence that the growth and abiotic stress resistance responses of halophyte T. halophila are
81 regulated by the key TF TsNAC1 in a dose-dependent manner.
82
83
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84 Results
85 TsNAC1 is an upstream regulator of TsVP1
86 A plasmid containing a 3× tandemly repeated 530 bp DNA fragment of the TsVP1 promoter (nt
87 -667 to -138) was constructed and introduced into the yeast strain Y187. This strain was then
88 transformed with the pGADT7-cDNA library comprising cDNAs from T. halophila plants
89 exposed to a 12-h high-salt stress treatment. One target protein was identified from the strains
90 grown on medium SD/-HIS/-TRP with 5 mM 3-AT. The candidate gene was cloned from T.
91 halophila, and the BLAST results indicated that the target protein was TsNAC1, a member of the
92 NAC superfamily. TsNAC1 has an ORF with a length of 918 bp, shares approximately 86%
93 similarity with RD26 at the nucleotide level and 92% similarity at the amino acid level, and
94 includes the A-E subdomains of the NAC DNA-binding domain (Fig. 1A). Evolutionary tree
95 analysis indicated that TsNAC1 is the closest gene to RD26 (ANAC072) in T. halophila
96 (Supplemental Fig. S1).
97 The coding region of TsNAC1 was fused to the GAL4 activation domain, and the promoter of
98 TsVP1 was cloned into the reporter plasmid pLacZi. Both of the plasmids were transformed into
99 the yeast strain YM4271. X-gal staining for β-galactosidase activity showed a blue color in strains
100 with the promoter region of TsVP1 (Fig. 1B), indicating that TsNAC1 was bound to the promoter
101 region to activate the transcription of TsVP1 in yeast.
102 The TsNAC1-GST fusion protein was expressed in E. coli, and the 130 bp fragments of the
103 TsVP1 promoter were labeled with digoxigenin-ddUTP for an electrophoretic mobility shift assay
104 (EMSA). TsNAC1 was bound to the labelled probe, and the 200× molar excess unlabeled probe
105 could competitively inhibit the combination (Fig. 1C). These results demonstrated that TsNAC1
106 could bind to the 130 bp sequence of the TsVP1 promoter in vitro.
107 Expression profile of TsNAC1
108 To evaluate the expression pattern of TsNAC1 in T. halophila, real-time RT-PCR was performed
109 with diverse organ samples collected from embryogenesis to senescence. The results suggested
110 that the senescent organ, including senescent cauline leaves, rosette leaves and roots, always had a
111 higher expression level than other stages. Parts of the floral organs, such as sepals and petals, also
112 exhibited a clear expression pattern (Supplemental Fig. S2A). These data were consistent with the
113 public microarray data for RD26 from the e-FP browser (Schmid et al., 2005).
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114 Plants (wild-type, WT) of T. halophila were sprayed and watered with 100 µM ABA or MeJA.
115 The roots were sensitive to ABA and showed a 600-fold expression peak after 24 h of treatment,
116 whereas the shoots were sensitive to MeJA and showed a 720-fold expression peak after 24 h of
117 treatment. Expression of TsNAC1 was also induced by cold, drought and active oxygen stress in
118 the shoots and by drought and salt stress in the roots (Supplemental Fig. S2B).
119 Overexpression of TsNAC1 retards the growth of T. halophila
120 Transgenic plants overexpressing TsNAC1 (OX, 35S:TsNAC1) and TsNAC1 RNAi plants (NR,
121 harboring hairpin-shaped TsNAC1 mRNA fragments (411-882 bp) driven by the CaMV 35S
122 promoter) were generated from T. halophila, and Arabidopsis overexpressing TsNAC1 plants
123 (OXA, 35S:TsNAC1) were also generated (Fig. 2A). Western blot analyses indicated that the
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124 protein levels of these independent lines were consistent with the transcription levels of TsNAC1
125 (Fig. 2C). The overexpressing lines OX7 and OX10 and RNAi lines NR10 and NR11 of T.
126 halophila were selected to determine the effects of TsNAC1 transcript levels on growth and
127 abiotic stress responses. Seeds were germinated on 1/2 MS-agar medium, and the overexpressing
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128 lines OX7 and OX10 seeds germinated later than the WT and RNAi seeds (Fig. 2B). After six
129 weeks of growth, the OX7 and OX10 plants were much smaller than the WT (Fig. 2B), and the
130 dry weights of the shoots and roots were substantially lighter (Fig. 2E). The dry weight of
131 6-week-old plants from the T. halophila OX lines was reduced by 10.1%-25.4% in the shoots and
132 by 49.5%-52.5% in the roots compared with those of the WT (Fig. 2E). In contrast, the NR10 and
133 NR11 plants had larger leaves and stronger roots (Fig. 2B). The biomass and growth rates were
134 inversely related to the expression levels of TsNAC1 (Fig. 2E). Taken together, these results led to
135 the conclusion that TsNAC1 retarded plant growth.
136 Overexpressing TsNAC1 improves abiotic stress resistance of plants
137 MeJA-responsive and ABA-responsive cis-acting regulatory elements are observed in the
138 promoter sequences of TsNAC1 and RD26, and the TsNAC1 promoter has an anaerobic induction
139 element and certain cold- and dehydration-responsiveness elements, including the TGA-element,
140 ARE and TC-rich repeats (Supplemental Tab. S1). To identify whether TsNAC1 expression
141 enhances plant tolerance to drought, high salinity, low temperatures, and reactive oxygen species
142 (ROS) stress in transgenic T. halophila, the seeds from the transgenic lines were germinated in a
143 medium of peat and vermiculite (at a 2:1 ratio), and the seedlings were grown for six weeks under
144 normal conditions. Under the low-temperature stress (-4℃) treatment (Fig. 2D), the
145 overexpressing plants had a higher survival rate after 48 h of treatment (Supplemental Fig. S3A).
146 After six weeks of growth, plants were watered with 18% PEG6000 solution for one week (Fig.
147 2D), and overexpressing plants exhibited higher tolerance to drought conditions than WT or RNAi
148 transgenic plants (Supplemental Fig. S3B). To examine salt resistance, the plants were thoroughly
149 watered with an 800 mM NaCl solution and then watered with 10 ml of a 400 mM NaCl solution
150 every 3 days (Fig. 2D). After 14 days of exposure, the transgenic RNAi and WT plants developed
151 necrotic leaves and certain senescent leaves died, whereas the TsNAC1 overexpression plants
152 displayed the same characteristics as untreated plants (Supplemental Fig. S3C). Uniform seedlings
153 of transgenic and WT plants were sprayed with 0.1 ml 3 mM paraquat per pot. After a 36 h
154 treatment the leaves of OX lines all showed greater resistance and higher survival probabilities
155 than the WT or NR plants (Supplemental Fig.S3D). These results suggested that overexpressing
156 TsNAC1 significantly improved plant abiotic resistance.
157 Exploration of the target genes of TsNAC1
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158 To investigate how the overexpression of TsNAC1 retarded plant vegetative development, the
159 downstream target genes of TsNAC1 were identified by ChIP-Seq using overexpressing lines OX7
160 and OX10 and the TsNAC1 polyclonal antibody, the specificity of which was tested by Western
161 blot, with Col-0 and OXA lines plants leaves as materials and with the β-Actin antibody as the
162 contrast (Supplemental Fig. S4). Each sequencing read was mapped to the T. halophila genome
163 (http://thellungiella.org/) to identify their position using the ultrafast Bowtie aligner (Langmead et
164 al., 2009). The ChIP-Seq data included 14.2 million mapped reads, with 7.8x genome coverage on
165 average. After excluding the peaks in intergenic regions, MACS software (Zhang et al., 2008)
166 detected 1868 and 2454 genes associated with a gene model with p-values of <0.05 for each of the
167 ChIP-Seq datasets. A total of 892 genes were highly enriched in both sets, and 22.16% of these
168 gene peaks were located in the promoter region (a fragment of from -2000 bp to ATG) (Fig. 3A).
169 Additionally, excepted the peaks in the intergenic, the reads distribution was close to the
170 transcription start site (TSS) at -200 bp to +100 bp (Fig. 3B). TsNAC1 is an upstream regulator of
171 TsVP1. The ChIP-Seq signals of TP1G13990 (TsVP1) and TP2G25870 (Tub2), which were used
172 as the positive and the negative controls, respectively, were visualized on the genome browser
173 (Fig. 3C). Based on the ChIP-Seq, motifs around the binding peaks located at the promoter
174 regions and a short and ungapped CA(T/A)G sequence were investigated in Figure 3D. The ChIP
175 samples were quantified via q-PCR using primers specific to the promoters of TsVP1 and Tub2. As
176 shown in Fig. 3E, the promoter region of TsVP1 was effectively enriched, and TsVP1 was
177 expressed at higher levels in OX lines than in the NR lines. These data demonstrated that TsNAC1
178 targeted the promoter of TsVP1 and the ChIP-Seq results were valid.
179 To further explore the biological processes regulated by TsNAC1, the binding target genes were
180 classified into gene ontology (GO) groups. The GO analysis results for 892 co-enriched genes
181 showed a significant and coincident enrichment for biological regulation (GO:0065007),
182 developmental processes (GO:0032502) and responses to stimuli (GO:0050896) compared with
183 the input sample data (Fig. 3F). The next hierarchical classification of these genes primarily
184 involved biological processes, including cell growth (GO:0016049), embryonic development
185 ending in seed dormancy (GO:0009793) and response to salt stress (GO:0009651) (Fig. 3F).
186 Genes with fold enrichment >20 (normalized by the input sample) and peaks located in the
187 promoter region of genes from these three groups were selected to test for the expression levels in
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188 transgenic plants, including the levels of TsVP1. Seven genes were up-regulated (log (OX/NR) >2)
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189 and 2 genes were down-regulated (log (OX/NR) <-1) by TsNAC1 (Tab. 1). The promoter regions
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190 for these nine genes were recombined into a pLacZi vector for one-hybrid yeast system assays of
191 the YM4271 strain. X-gal staining showed that these regions were targeted by TsNAC1 in yeast
192 (Fig. 4A).
193 TsNAC1 regulated genes with the CA(T/A)G element in the promoter
194 Motif discovery with the DNA fragments identified via ChIP-Seq around the binding peaks was
195 performed using MEME-ChIP (Bailey et al., 2015), and 197 DNA sequences (length between 67
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196 to 434, average length 151.1) located at the promoter regions and a short and ungapped CA(T/A)G
197 motif with an E-value of <8.5e-018 were investigated (Fig. 3D). The distribution of the motif was
198 visualized using a histogram that showed the percent occurrence of the CA(T/A)G motif (x axis)
199 plotted against the log2-fold enrichment (FC) of IP samples compared with the input sample (y
200 axis), with the color of each square mapped to the number of indicated peak motifs. (Fig. 3D). In
201 general, peaks had one or more motifs at log2 >0, and the proportion of peaks containing a motif
202 at least increased along with log2 FC of TsNAC1 binding.
203 It had been demonstrated that TsNAC1 could recognize the 130 bp key region of the promoter of
204 TsVP1 in vivo and vitro. To identify the specific binding site, a serious 5`-deletion fragment was
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