BpNAC012 positively regulates abiotic stress responses and secondary wall biosynthesis

69 Pages·2017·3.07 MB·English

Plant Physiology Preview. Published on December 10, 2018, as DOI:10.1104/pp.18.01167

1 Title page
2
3 BpNAC012 positively regulates abiotic stress responses and secondary
4 wall biosynthesis1
5
6 Ping Hu1, Kaimin Zhang1, Chuanping Yang1,*
7 1State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University), 26
8 Hexing Road, 150040 Harbin, China
9 *Corresponding author: [email protected]
10
11 Running Title: Functional roles of BpNAC012 in Betula platyphylla
12
13 One-sentence Summary: BpNAC012 is a transcriptional regulator of both abiotic stress
14 responses and secondary wall biosynthesis.

Author Contributions: Y.C.P. and H.P. conceived the original screening and research
plans; H.P. performed most of the experiments and analyzed the data; Z.K.M. provided
technical assistance to H.P.; H.P. conceived the project and wrote the article with
contributions of all the authors; Y.C.P. supervised and complemented the writing.

11
Downloaded from on April 6, 2019 - Published by www.plantphysiol.org
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
Copyright 2018 by the American Society of Plant Biologists
15 ABSTRACT
16 NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in plant
17 biological processes and stress responses. Here, we characterized the functional roles of
18 BpNAC012 in white birch (Betula platyphylla). We found that BpNAC012 serves as a
19 transcriptional activator. Gain- and loss-of-function analysis revealed that the transcript
20 level of BpNAC012 was positively associated with salt and osmotic stress tolerance.
21 BpNAC012 activated the core sequence CGT[G/A] to induce the expression of abiotic
22 stress-responsive downstream genes, including P5CS1/P5CS2 (for delta
23 1-pyrroline-5-carboxylate synthetase), SODs (for superoxide dismutase), and PODs (for
24 peroxidase), resulting in enhanced salt and osmotic stress tolerance in BpNAC012
25 overexpression transgenic birch lines. We also showed that BpNAC012 is predominately
26 expressed in mature stems and that RNA interference (RNAi)-induced suppression of
27 BpNAC012 caused a drastic reduction in the secondary wall thickening of stem fibers.
28 Overexpression of BpNAC012 activated the expression of secondary wall-associated
29 downstream genes by directly binding to the SNBE (secondary-wall NAC binding
30 element) sites, resulting in ectopic secondary wall deposition in the stem epidermis.
31 Moreover, salt and osmotic stresses elicited higher expression levels of lignin
32 biosynthetic genes and elevated lignin accumulation in BpNAC012 overexpression lines.
33 These findings provide insight into the functions of NAC transcription factors.
34
35 Key-words: BpNAC012, Betula platyphylla, abiotic stress responses, secondary wall
2
Downloaded from on April 6, 2019 - Published by www.plantphysiol.org
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
36 biosynthesis, gene expression regulation, cis-acting elements
37 INTRODUCTION
38 Plant-specific NAM (no apical meristem), ATAF1/2, and CUC2 (cup-shaped
39 cotyledon) (NAC) proteins belong to one of the largest family of transcription factors,
40 which are expressed in different tissues at various developmental stages. The number of
41 NAC members in different plant species varies greatly, from 30 in the early divergent
42 land plants to 177 genes in soybean (Glycine max) (Zhu et al., 2012; Cenci et al., 2014).
43 To date, 105 NAC genes have been identified in Arabidopsis thaliana, 113 in sorghum
44 (Sorghum bicolor), 115 in maize (Zea mays), 138 in rice (Oryza sativa), and 163 in
45 poplar (Populus trichocarpa) (Voitsik et al., 2013; You et al., 2015). All NAC family
46 proteins contain a conserved NAC domain with a stretch of approximately 150
47 conserved amino acids at the N-terminus, which serves as a platform for DNA binding
48 and is also responsible for the formation of homo- or hetero-dimers with other NAC
49 domain proteins (Lindemose et al., 2014; Ooka et al., 2003; Welner et al., 2012; Fang et
50 al., 2014). The C-terminal regions of NAC proteins commonly contain simple amino
51 acid repeats, including the repeats of proline and glutamine, serine and threonine, or
52 acidic residues (Olsen et al., 2005). The C-terminal region of NAC proteins serves as a
53 transcriptional activator or transcriptional repressor, which is a diversified domain with
54 variable sequence and length (Xie et al., 2000; Hao et al., 2011). As important regulatory
55 proteins, transcription factors function by binding to cis-elements in the promoters of
56 their target genes. The motifs bound by NAC proteins have been studied.
3
Downloaded from on April 6, 2019 - Published by www.plantphysiol.org
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
57 ANAC019/055/072 specifically bind to the core DNA motif CACG (Tran et al., 2004;
58 Zhou et al., 2013). Additionally, NAC proteins specifically bind to a DNA motif
59 TTNCGT[G/A], but with different affinities (Jensen et al., 2010). The target promoter
60 regions bound by NAC proteins mostly contain the core sequence CGT[G/A] (Olsen et
61 al., 2005; Xu et al., 2013; Lindemose et al., 2014). Secondary wall NAC master switches
62 bind to a common cis-element, named as secondary-wall NAC binding element (SNBE),
63 which is composed of an imperfect palindrome of 19-bp with consensus sequence
64 (T/A)NN(C/T)(T/C/G)TNNNNNNNA(A/C)GN(A/C/T)(A/T) (Zhong et al., 2010a,b).
65 Plant NAC proteins play important roles in diverse processes, such as control of
66 boundary cell formation (Aida et al., 1997), lateral root development (Xie et al., 2000),
67 leaf senescence (Guo and Gan, 2006), flowering (Ying et al., 2014; Kim et al., 2007),
68 boundary and shoot meristem formation (Vroemen et al., 2003), secondary cell wall
69 synthesis (Mitsuda et al., 2005, 2007; Zhong et al., 2006) and xylem vessel
70 differentiation (Kubo et al., 2005; Yamaguchi et al., 2011). Overexpression of NAC
71 domain genes PtVNS (for VND-, NST/SND-, and SMB-related proteins)/PtrWND (for
72 wood-associated NAC domain transcription factor) from poplar (Populus trichocarpa)
73 induced ectopic secondary wall thickening in poplar leaves, suggesting that wood
74 formation in poplar is controlled by cooperative functions of the NAC proteins (Ohtani
75 et al., 2011). PtrWND2B and PtrWND6B activate the promoter activities of a number of
76 wood-associated transcription factors and wood biosynthetic genes in poplar, suggesting
77 that PtrWNDs, and their downstream transcription factors, form a transcriptional
4
Downloaded from on April 6, 2019 - Published by www.plantphysiol.org
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
78 network to regulate wood formation in poplar (Zhong et al., 2010a). Moreover, NAC
79 proteins also play important roles in plant responses to abiotic stresses. A rice
80 stress-responsive NAC gene (SNAC1) is highly induced by osmotic stress, and can
81 improve drought and salt tolerance by enhancing root development and reducing
82 transpiration rates (Liu et al., 2014). SNAC1 also activates the expression of genes
83 involved in abiotic stresses and abscisic acid (ABA) signaling, including sucrose
84 phosphate synthase, 1-phosphatidylinositol-3-phosphate-5-kinase, regulatory
85 components of ABA receptor and type 2C protein phosphatases (Saad et al., 2013). A
86 rice NAC protein (OsNAP) enhances salinity and drought tolerance by inducing the
87 expression of OsPP2C06/OsABI2, OsPP2C09, OsPP2C68, and OsSalT, and
88 transcription factors involved in stress (OsDREB1A, OsMYB2, OsAP37 and OsAP59)
89 (Chen et al., 2014). Overexpression of TaNAC2, a NAC member from wheat (Triticum
90 aestivum), enhances tolerances to drought, salt, and freezing stresses in transgenic
91 Arabidopsis lines by inducing the expression of abiotic stress-responsive genes DREB2A,
92 RD22, ABI2, and ABI5 (Mao et al., 2012).
93 Although many NACs have been cloned and identified in various plant species, only a
94 few of them have been characterized functionally (You et al., 2015). White birch (Betula
95 platyphylla) is one of the main broad-leave tree species with a characteristic of fast
96 growth. This widely grown tree is tolerant to cold and drought, and has important
97 applications in biofuels and pulp industries (Li et al., 2002; Borrega et al., 2013). In the
98 present study, we characterized the function of BpNAC012, a NAC transcription factor
5
Downloaded from on April 6, 2019 - Published by www.plantphysiol.org
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
99 from the Japanese white birch. We found distinct roles of BpNAC012 in regulating
100 abiotic stress responses and secondary wall biosynthesis. Chromatin
101 immunoprecipitation (ChIP) assays revealed the binding specificity of BpNAC012 to the
102 core sequence CGT[G/A] and the SNBE site in the promoters of abiotic
103 stress-responsive downstream genes and secondary wall-associated downstream genes,
104 respectively. This novel mechanism may be relevant to other transcription factors that
105 generate specific patterns of gene expression in plant development and stress responses.
106
107 RESULTS
108 Cloning and sequence analysis of BpNAC012
109 Previously, we generated transcriptomes of birch, and a NAC homolog that was highly
110 expressed in birch stems was identified and investigated in this study (Wang et al., 2014).
111 The coding sequence (CDS) of this NAC gene is 1281 bp, encoding a deduced protein of
112 426 amino acids. The phylogenetic tree was constructed for this birch NAC gene with 24
113 Arabidopsis NAC genes that are representative of different NAC subfamilies by the
114 neighbor-joining method using MEGA5. The result revealed that this birch NAC gene is
115 closely related to the Arabidopsis SND1 gene (AT1G32770, also called NST3/ANAC012)
116 (Supplemental Fig. S1). Therefore, we designated the birch NAC gene as BpNAC012,
117 which was deposited in GenBank with accession number KT344119.
118 The expression patterns of BpNAC012
119 The transcript level of BpNAC012 was determined using reverse transcription
6
Downloaded from on April 6, 2019 - Published by www.plantphysiol.org
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
120 quantitative PCR (RT-qPCR) in different tissues. The transcript level of BpNAC012 is
121 highest in stems, followed by the leaves and apical buds; BpNAC012 expression is
122 lowest in the roots, which was used as a calibrator to normalize the expression level in
123 other tissues (Fig. 1A). These results indicated that BpNAC012 has a tissue-specific
124 expression profile, and is predominately expressed in stems. Additionally, BpNAC012 is
125 preferentially expressed in mature stems or leaves rather than in young stems or leaves,
126 indicating that BpNAC012 is also involved in plant development.
127 The expression of BpNAC012 in birch leaves in response to salt, osmotic, and drought
128 stresses or ABA treatment was further studied. The results showed that the expression of
129 BpNAC012 is induced by both NaCl and mannitol treatments, and reached peak levels at
130 24 h of stress (Fig. 1B). We further studied the expression of BpNAC012 in the leaves of
131 birch plants grown in soil without water for 14 d. The results showed that the expression
132 of BpNAC012 increased from 3 to 14 d without watering (Fig. 1B). Under ABA
133 treatment, the expression of BpNAC012 was induced and peaked at 9 h (Fig. 1B). These
134 results indicated that BpNAC012 expression responds to salt, osmotic, and drought
135 stresses, suggesting an involvement in abiotic stress responses.
136 Determination of the transcriptional activation domain of BpNAC012
137 To determine the transcriptional activation domain of BpNAC012, the full-length or
138 truncated CDS of BpNAC012 was fused with the GAL4 DNA-binding domain and
139 transformed into Y2H Gold Yeast Strain (Clontech) for transcriptional activity detection.
140 The full-length CDS demonstrated transactivation ability, indicating that BpNAC012 is a
7
Downloaded from on April 6, 2019 - Published by www.plantphysiol.org
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
141 transcriptional activator (Supplemental Fig. S2). Additionally, the minimal truncated
142 region that showed transcriptional activity is from amino acid 145 to 215; further
143 deletion of this region completely abolished the transactivation activity (Supplemental
144 Fig. S2), suggesting that the transcriptional activation domain of BpNAC012 is in amino
145 acids 145–215.
146 Generation of transgenic birch with overexpression or suppression of BpNAC012
147 To investigate the function of BpNAC012 using gain- and loss-of-function methods,
148 transgenic birch plants that overexpressed BpNAC012 or had BpNAC012 suppressed by
149 RNA interference (RNAi) were generated using Agrobacterium-mediated transformation.
150 16 lines overexpressing BpNAC012 (OE) and 17 lines with RNAi-suppressed BpNAC012
151 (RS) were generated. The expression levels of BpNAC012 in the transgenic lines were
152 studied using RT-qPCR (Supplemental Fig. S3). Two BpNAC012 overexpression lines,
153 OE3 and OE8, were randomly selected for gain-of-function study; two
154 RNAi-suppression BpNAC012 transgenic lines, RS1 and RS6, where the expression of
155 BpNAC012 was greatly decreased, were selected for loss-of-function analysis
156 (Supplemental Fig. S3).
157 Salt and osmotic stress tolerance test
158 The NaCl- and mannitol-inducible expression of BpNAC012 prompted us to analyze
159 its potential role in salt and osmotic resistance (Fig. 1B). The plantlets of
160 BpNAC012-OE, wild type (WT), and BpNAC012-RS of similar sizes were cultured on
161 1/2MS medium containing 80 mM NaCl or 100 mM mannitol for 21 d, and plantlets
8
Downloaded from on April 6, 2019 - Published by www.plantphysiol.org
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
162 grown on 1/2MS medium were used as a control. Under normal conditions, there was no
163 difference in the growth and phenotype among these lines. Under NaCl and mannitol
164 stress conditions, the transgenic OE plants grew better than the WT and RS plantlets and
165 remained green; however, the RS lines wilted and died (Fig. 2A). Additionally, the fresh
166 weight and root length of the OE lines were the highest, followed by the WT plants; the
167 RS lines had the lowest fresh weight and root length (Fig. 2, B and C). These results
168 indicated that overexpression of BpNAC012 confers substantially improved tolerance to
169 salt and osmotic stresses in transgenic plants.
170 Proline biosynthesis is regulated by BpNAC012
171 The proline content was measured among the BpNAC012-OE, WT, and
172 BpNAC012-RS plants. Under normal conditions, each studied line had similar proline
173 content. Under salt and osmotic stress treatments, all lines displayed increased proline
174 contents relative to that under normal conditions. Additionally, the OE lines accumulated
175 significantly higher proline contents, and the RS lines exhibited significantly decreased
176 proline contents compared with WT plants (Fig. 2D).
177 As the proline contents were altered in the different birch lines, we further examined
178 the expression of two birch proline biosynthesis-related genes, BpP5CS1 and BpP5CS2.
179 Under normal conditions, there was no difference in the expression levels of BpP5CS1
180 and BpP5CS2 among the studied lines. Under salt and osmotic stress conditions, the
181 expression levels of BpP5CS1 and BpP5CS2 significantly increased in the OE lines, but
182 were significantly reduced in the RS lines compared with the WT plants (Fig. 2, E and F).
9
Downloaded from on April 6, 2019 - Published by www.plantphysiol.org
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
183 These results indicated that BpNAC012 can regulate the biosynthesis of proline in
184 response to salt and osmotic stresses.
185 Reactive oxygen species (ROS) scavenging assay
186 H O and O2-, two prominent ROS species, were determined by DAB
2 2
187 (3,3’-diaminobenzidine) and NBT (nitroblue tetrazolium) in situ staining, respectively,
188 which were visualized as deep brown and dark blue products, respectively. Similar
189 results were observed in two OE lines or in two RS lines, and representative images are
190 shown in Figure 3A and Figure 3B. H O and O2- were largely reduced in leaves of the
2 2
191 two OE lines compared with the WT plants under salt or osmotic stress conditions (Fig.
192 3, A and B). Meanwhile, H O and O2- in RS1 and RS6 lines were substantially higher
2 2
193 than that in the WT plants (Fig. 3, A and B). Measurement of H O also confirmed that
2 2
194 both RS lines had the highest H O content, followed by the WT, and the OE lines had
2 2
195 the lowest H O levels (Fig. 3C), which was consistent with the DAB staining.
2 2
196 We further studied whether the altered ROS level reflects a changed ROS scavenging
197 capability in the plants. The activities of superoxide dismutase (SOD) and peroxidase
198 (POD), the two main antioxidant enzymes involved in ROS scavenging, were
199 determined. Under normal conditions, there was no difference in SOD and POD
200 activities among the BpNAC012-OE, WT, and BpNAC012-RS lines (Fig. 3, D and E).
201 Under NaCl and mannitol treatments, both SOD and POD activities were significantly
202 enhanced in the OE lines, but were reduced in the RS lines compared with the WT plants
203 (Fig. 3, D and E).
10
Downloaded from on April 6, 2019 - Published by www.plantphysiol.org
Copyright © 2018 American Society of Plant Biologists. All rights reserved.