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Plant Science journal homepage: www.elsevier.com/locate/plantsci
WRKY13 acts in stem development in Arabidopsis thaliana
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Wei Li a,b , Zhaoxia Tian a , Diqiu Yu b,∗ a
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School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, Yunnan 666303, China
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Article history: Received 19 October 2014 Received in revised form 23 March 2015 Accepted 3 April 2015 Available online xxx
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Keywords: WRKY13 Weaker stems Sclerenchyma Lignin Arabidopsis thaliana
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1. Introduction
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Stems are important for plants to grow erectly. In stems, sclerenchyma cells must develop secondary cell walls to provide plants with physical support. The secondary cell walls are mainly composed of lignin, xylan and cellulose. Deficiency of overall stem development could cause weakened stems. Here we prove that WRKY13 acts in stem development. The wrky13 mutants take on a weaker stem phenotype. The number of sclerenchyma cells, stem diameter and the number of vascular bundles were reduced in wrky13 mutants. Lignin-synthesis-related genes were repressed in wrky13 mutants. Chromatin immunoprecipitation assays proved that WRKY13 could directly bind to the promoter of NST2. Taken together, we proposed that WRKY13 affected the overall development of stem. Identification of the role of WRKY13 may help to resolve agricultural problems caused by weaker stems. © 2015 Elsevier Ireland Ltd. All rights reserved.
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Despite the organ shape varying in plant species (such as leaves and flowers), many dicotyledonous plants have similar stem structures, which are comprised of epidermis, cortex, vascular bundles, interfascicular tissues, and pith. Unlike parenchyma cells in pith, cells in vascular bundles and interfascicular tissues are sclerenchyma with two kinds of cell walls, primary and secondarily-thickened. Secondary walls are deposited inside the primary walls after cells cease their expansion [1]. The secondary cell walls in plants can be viewed as thermochemical energy storage systems and are considered as an important renewable source of bioenergy [2,3]. Secondary walls also provide mechanical strength to the stem and enable vascular plants to withstand transpirationcaused negative pressure in their vessels [4]. Lignin is an aromatic polymer deposited in secondary thickened cells where it provides strength and impermeability to the
Abbreviations: SND1, secondary wall-associated NAC domain 1; NST1, NAC secondary wall thickening promoting factor1; GUS, -glucuronidase; X-gluc, 5-bromo-4-chloro-3-indolyl b-d-glucuronic acid; ChIP, chromatin immunoprecipitation; PAL4, phenylalanine ammonia-lyase 4; 4CL1, 4-coumarate:coa ligase 1; CesA7, cellulose synthase catalytic subunit 7; CesA8, cellulose synthase 8; CCoAOMT1, caffeoyl coenzyme A O-methyltransferase 1; CAD-c, cinnamyl alcohol dehydrogenase; F5H, ferulate 5-hydroxylase; HCT, hydroxycinnamoyl transferase; CCR1, cinnamoyl CoA reductase 1. ∗ Corresponding author. Tel.: +86 871 5178133; fax: +86 871 5160916. E-mail address: [email protected] (D. Yu).
wall [5]. In dicot plants, the formation of lignin is controlled by many enzymes, such as: phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (4CL) and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) [6]. Besides lignin, the secondary cell wall is composed of many other components. To ensure proper growth, biosynthesis of these cell wall components is highly coordinated via a cascade of transcription factors. Several transcription factors, including the NAC and MYB families, have been implicated in regulating cell wall biosynthesis or deposition. Two NAC genes, NAC secondary wall thickening promoting factor1 (NST1) and NST2, act redundantly in secondary wall thickening in the endothecium [7]. Disruption of MYB26 can cause failure of secondary wall thickening in anther endothecium [8]. In Arabidopsis thaliana, the WRKY transcription factor superfamily has 74 members with diverse functions [9,10]. For example, WRKY57 functions as a node of convergence for JA- and auxinmediated signals in leaf senescence [11]. wrky8 mutant plants are even more hypersensitive to salt and susceptible to cruciferinfecting tobacco mosaic virus infection [12,13]. Disruption of WRKY12 initiates pith secondary wall formation and increases stem biomass in dicotyledonous plants [14]. In this report, we provide evidence that mutation of WRKY13 leads to a weaker stem phenotype in A. thaliana, and WRKY13 is a positive regulator for overall stem development. Stem diameter and the number of vascular bundles are significantly decreased in wkry13 mutants. WRKY13 upregulates transcript levels of lignin pathway genes and lignin content in stem. In addition, the secondary cell wall synthesis gene NST2 is proved to be the direct downstream target of WRKY13.
http://dx.doi.org/10.1016/j.plantsci.2015.04.004 0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: W. Li, et al., WRKY13 acts in stem development in Arabidopsis thaliana, Plant Sci. (2015), http://dx.doi.org/10.1016/j.plantsci.2015.04.004
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2. Materials and methods
2.6. ChIP assays
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2.1. Plant growth conditions
Chromatin immunoprecipitation (ChIP) assays were performed essentially as previously described [17,18]. Inflorescence stems of wild type (WT) and wrky13-1/PW13:W13 L6 transgenic plants were used for ChIP assays. HA antibody (Thermo Fisher Pierce, Rockford, IL, USA) was used to immunoprecipitate protein–DNA complex, and the precipitated DNA was purified by using a PCR purification kit for real-time quantitative-PCR analysis. The ACTIN7 (AT5G09810) 5 untranslated region sequence was used as an endogenous control. The relative quantity value is presented as the DNA binding ratio (differential site occupancy). The primers used for different promoters are listed in Supplementary Table S1.
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A. thaliana (accession Columbia) seeds (obtained from the Arabidopsis Information Resource, http://www.arabidopsis.org) were surface sterilized with 20% bleach and washed three times with sterile water. Sterilized seeds were vernalized in darkness for 2 days at 4 ◦ C, planted in soil, and then grown in a tissue culture room at 22 ◦ C and 75% humidity under a 16 h-light/8 h-dark photoperiod. 2.2. Expression analysis For real-time RT-PCR analysis, total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I (Fermentas, Vilnius, Lithuania) according to the manufacturer’s instructions. Total RNA (∼2 g) was reversetranscribed in a 20-L reaction mixture by using Superscript II (Invitrogen). After that, 1-L aliquots were used as templates for real-time RT-PCR. Reactions (20 L) were performed with a Lightcycler FastStart DNA Master SYBR Green I Kit (Roche, Penzberg, Germany) in a Roche LightCycler 480 real-time PCR machine, according to the manufacturer’s instructions. ACTIN2 (AT3G18780) was used as a control, and three biological replicates were conducted. The primers used for real-time RT-PCR amplification of different genes are listed in Supplementary Table S1. For -glucuronidase (GUS) staining, histochemical detection of GUS activity was performed with 5-bromo-4-chloro-3-indolyl b-d-glucuronic acid (X-gluc) as the substrate. Plant tissues were prefixed in ice-cold 90% (vol/vol) acetone for 20 min, and then washed three times with GUS staining buffer (without X-gluc) before incubation in X-gluc solution [1 mM X-gluc, 50 mM NaPO4 (pH 7), 1 mM K3 Fe(CN)6 , 1 mM K4 Fe(CN)6 , and 0.05% Triton X-100] under a vacuum for 10 min at room temperature and then incubated overnight at 37 ◦ C. Several changes of 70% (vol/vol) ethanol were used to remove the chlorophyll. 2.3. Measurement of breaking force The basal parts of 6-week-old inflorescence stems were measured. The force needed to pull samples apart was measured through dynamic mechanical analysis with a DMA Q800 (TA Instruments, New Castle, DE, USA). Ends of each stem segment were clamped at the same distance and torn apart at the same speed. 2.4. Tissue sections and microscopy analysis
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Stem samples were fixed in FAA buffer (formaldehyde:glacial acetic acid:50% ethanol, 1:1:18), and then embedded in paraffin. Specimens were cut into 8-m-thick sections and stained with 0.05% toluidine blue O and observed under Zeiss Axio Scope (Zeiss, Oberkochen, Germany).
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2.5. Lignin measurements
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2.7. GUS activity assays Constructs were introduced into Agrobacterium tumefaciens (strain GV3101), and infiltration of Nicotiana benthamiana was performed as described previously [12]. Infected tissues were analyzed for 48 h after infiltration. GUS activity was measured as previously described [19] except for extracts which were not first purified by column chromatography.
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Thioglycolic acid (TGA) assay was carried out according to Campbell and Ellis [15] and Jones et al. [16]. The crude cell-wall preparations were saponified in 1 M NaOH before being extracted through TGA for 3 h at 80 ◦ C. The insoluble material was collected by centrifugation, washed with distilled water, incubated in 1 M NaOH overnight and set on a rotating shaker at room temperature. The supernatant was transferred with 200 mL concentrated HCl added. The precipitate was collected and resuspended in 1 M NaOH. The OD280 was read under a tenfold sample dilution.
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3. Results
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3.1. The wrky13 mutants take on a weaker stem phenotype
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We screened several transfer (T)-DNA insertion mutants obtained from the Arabidopsis Biological Resource Center for mutants of the stem phenotype. Two T-DNA insertion mutants, wrky13-1 (SALK 064346C) and wrky13-2 (SALK 032912) [20], were isolated. These two lines were predicted to have T-DNA insertions in the AtWRKY13 (AT4G39410) gene (Fig. 1A). Then, RNA of the two mutant plants was extracted and subjected to RT-PCR. No full-length transcript was detected in either wkry13-1 or wrky13-2 (Fig. 1B). Under long-day conditions, mutation in WRKY13 did not lead to obvious developmental problem during the seedling stage (Fig. 1C). However, in mature plants, wrky13-1 and wrky13-2 displayed a weaker stem phenotype, while other organs grew normally (Fig. 1C). To ascertain the function of WRKY13 in stems, we fused HA tag and WRKY13 CDS into the same expression vector driven by the WRKY13 promoter (Supplementary Fig. S1A). The construct was transformed into wrky13-1 to generate wrky13-1/PW13:W13. Transcripts of WRKY13 were detected in transgenic plants (Supplementary Fig. S1B). WRKY13 was sufficient to rescue the weaker stem phenotype of wkry13-1 (Supplementary Fig. S1C). We wondered if this weaker stem phenotype of wrky13-1 indicated that the mechanical strength necessary to support the plant was lower in mutant plants. As shown in Fig. 1D, the forces needed to pull wrky13-1 mutant stems apart decreased sharply, while the breaking force for the wrky13-1/PW13:W13 transgenic stems was similar to what required for WT. 3.2. Expression analysis of WRKY13
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Since wrky13 mutant stems developed weaker stem phenotype, we investigated the expression pattern of WRKY13, especially in stems. RNA extracted from different organs of WT plants was used to perform real-time RT-PCR. We found that WRKY13 was preferentially expressed in stem internodes, where its transcript level increased with maturity (Fig. 2A). WRKY13 gene transcripts were also detected in roots. To further elucidate the expression pattern of WRKY13, we fused a 2.5 kb promoter of WRKY13 to a -glucuronidase (GUS) reporter. The construct was used to transform WT Arabidopsis. Several lines
Please cite this article in press as: W. Li, et al., WRKY13 acts in stem development in Arabidopsis thaliana, Plant Sci. (2015), http://dx.doi.org/10.1016/j.plantsci.2015.04.004
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Fig. 1. wrky13 mutants developed weaker stem phenotype. (A) Characterization of WRKY13 (AT4G39410) gene. The exons, introns and untranslated regions are represented by black boxes, black lines and white boxes, respectively. The T-DNA insertion lines are designated wrky13-1 (Salk 064346C) and wrky13-2 (Salk 032912) with insertions in exons. A pair of primers (W13F and W13R) are indicated as arrows. (B) Aaerial part of 5-week-old plants were harvested for RNA extraction. Primers W13F and W13R were used to determine WRKY13 transcript levels. ACTIN2 was used as an internal control. (C) wrky13 mutants developed weaker stems compared to that of WT. Plants were grown under long-day conditions. Scale bars represent 1 cm. (D) Less force is needed to break wrky13 mutant inflorescence stems. The basal part of inflorescence stem of 6-week-old plants were analyzed. Error bars represent ±SD from 15 independent experiments. **P < 0.001, Student’s t test.
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Fig. 2. Expression pattern of WRKY13. (A) Long-day grown WT plants were used for RNA extraction. Quantitative RT-PCR showing expression of WRKY13 in different organs normalized to expression of ACTIN2. Error bars represent ±SD from 3 independent experiments. *P < 0.05; **P < 0.001, Student’s t test. (B–E) Expression patterns determined by the WKRY13 promoter--glucuronidase (GUS) construct. At least 5 independent transgenic lines showed similar expression pattern. (F) Cross section of WKRY13 promoterGUS transgenic plant stem. The section is 8 m thick. GUS activity is detected in secondary wall-containing cell types. (G) High magnification showing the detailed expression in stem sections. Scale bars in (F) and (G) represent 50 m.
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Fig. 3. The development of sclerenchyma cells was repressed in wrky13 mutants. (A and B) Cross-sections of the basal part of 6-week-old inflorescence stems of wild type (A) and wrky13-1 (B) were subjected to toluidine blue O staining. (C and D) Enlarged portion of A and B, respectively. ph, phloem; xy, xylem; if, interfascicular fibers. Scale bars represent 100 m. (E) Schematic representation of methods used in (H)–(J). The red circle indicates cross sectional area of entire stem. The area within two yellow circles represents sclerenchyma cell area. (F–J) Quantitative statistics of stem cross-sections. Error bars represent ±SD from 8 independent experiments. *P < 0.05; **P < 0.001, Student’s t test.
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of transgenic plants showed similar results and confirmed the preferential expression in inflorescence stems (Fig. 2B). GUS signals were also detected in leaf vein, root and base of pod (Fig. 2C–E). In addition, we cut thin cross-sections of GUS transgenic plants to investigate the expression pattern of WRKY13 in stem. As shown in Fig. 2F and G, GUS activities were detected in all the cells in the stele. This indicates that WRKY13 may function in stem development. 3.3. The development of sclerenchyma is repressed in wrky13 mutants To investigate the anatomical defects of mutants, thin crosssections of stems were cut and subjected to toluidine blue O staining. As shown in Fig. 3, the stem diameter decreased significantly from 890 ± 25 m for WT (Fig. 3A) to 676 ± 19 m and 674 ± 20 m for wrky13-1 (Fig. 3B) and wrky13-2 (Supplementary Fig. S2A and Fig. 3F), respectively. The stem diameter of wrky131/PW13:W13 L6 (Supplementary Fig. S2B) is 894 ± 27 m, which was similar to that of WT (Fig. 3F). The number of vascular bundles decreased from 6.3 ± 0.4 for WT to 4.2 ± 0.2 and 4.6 ± 0.3 for wrky13-1 and wrky13-2 plants, respectively (Fig. 3G). There was no significant difference in the number of vascular bundles between WT and wrky13-1/PW13:W13 L6 (Fig. 3G). In addition, we measured cross-sectional area of the entire inflorescence stem and cross-sectional area of the area containing sclerenchyma cells (Fig. 3E). The cross-sectional area of the entire stem decreased from 128 ± 6.1 and 123 ± 6.4 inch2 for WT and wrky13-1/PW13:W13 L6 to 74 ± 5.5 and 74 ± 5.8 inch2 for wrky13-1 and wrky13-2, respectively (Fig. 3H). The cross-sectional area containing sclerenchyma cells decreased from 50 ± 2.1 and 46 ± 1.8 inch2 for WT and wrky131/PW13:W13 L6 to 21 ± 1.8 and 23 ± 1.7 inch2 for wrky13-1 and wrky13-2, respectively (Fig. 3I). Because mechanical support for the plant is derived mainly from sclerenchyma cells, the ratio of
sclerenchyma cell area divided by cross-sectional stem area could reflect stem mechanical strength. Again, these ratios decreased from 39 ± 1.4 and 37 ± 1.3% for WT and wrky13-1/PW13:W13 L6 to 28 ± 1.1 and 31 ± 1.2% for wrky13-1 and wrky13-2, respectively (Fig. 3J). The defect in sclerenchyma development was further confirmed by staining for lignin with phloroglucinol HCl (Fig. 4A–C). To confirm the reduction in the lignin content of wrky13 stems, thioglycolic acid assays were carried out for stem material. Fig. 4D demonstrates that the lignin content decreased in wrky13 stems. These observations confirmed the sclerenchyma development defects in wrky13 mutants. This conclusion is consistent with the function of sclerenchyma in providing physical support for plants to grow erectly.
3.4. Mutation of WRKY13 alters the expression level of several genes Since the lignin content decreased in wrky13 stems. We wondered if levels of lignin pathway genes were affected by the mutation of WKRY13. Real-time RT-PCR analysis was used to examine gene transcript levels. As shown in Fig. 5A, levels of PAL4 (AT3G10340), 4CL1 (AT1G51680), HCT (AT5G48930), and CAD6 (AT4G37970) were reduced, while CCoAOMT1 (AT4G34050), CCoAOMT7 (AT4G26220), CAD-c (AT3G19450), F5H (AT4G36220), and CCR1 (AT1G15950) remained unchanged in wrky13 stems. We also examined the levels of transcription factors that are related to sclerenchyma secondary wall synthesis [21–24]. The results showed that SND1(AT1G32770), NST1(AT2G46770), NST2 (AT3G61910), CesA7 (AT5G17420), and CesA8 (AT4G18780) were repressed by the disruption of WRKY13. Other genes, such as, C3H14 (AT1G66810) and C3H14L (AT1G68200), were not significantly changed in wrky13 mutants.
Fig. 4. The biosynthesis of lignin was affected in wrky13 mutants. (A-C) Free-hand sections of 6-week-old inflorescence stems were subjected to phloroglucinol-HCl staining. Red area represent lignin. (A) Wild type; (B) wrky13-1; (C) wrky13-2. Scale bars represent 100 m. (D) Thioglycolic acid (TGA) assays were carried out on stem material of WT and mutants. 6-week-old inflorescence stems were used for the TGA assays. Error bars represent ±SD from 8 independent experiments. *P < 0.05, Student’s t test.
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3.5. WRKY13 binds to the NST2 promoter Previous reports indicated that WRKY transcription factors specifically bind to W-box elements containing a TGAC core sequence in their target gene promoters [25]. To investigate the relationship between WRKY13 and its downstream targets, we searched promoters of several genes for W-box and performed in vivo chromatin immunoprecipitation (ChIP) assays by using wkry13-1/PW13:W13 L6 plants. Detailed ChIP analysis indicated that WRKY13 bound to the promoter regions of NST2 (Fig. 6A–C). We did not observe any specific binding of WRKY13 to the tested PAL4, 4CL1, CESA7 and CESA8 promoters (Supplementary Fig. S3). To further confirm the positive regulatory function of WRKY13, we performed transient expression assays. A reporter plasmid was constructed by fusing a 2-kb promoter sequence of NST2 with the -glucuronidase (GUS) reporter gene. The effector plasmid had a WRKY13 or GFP gene driven by the cauliflower mosaic virus (CaMV) 35S promoter. As shown in Fig. 6D, coexpression of the WRKY13 gene produced significantly higher GUS activity than coexpression of the GFP gene. This supported the hypothesis that WRKY13 could directly activate the expression of NST2.
4. Discussion
Fig. 5. Lignin pathway genes and secondary-wall-related genes are altered in wrky13 mutants. (A and B) Stems of 5-week-old WT plants were harvested for RNA extraction. Quantitative RT-PCR was used to analysis genes expression levels. Error bars represent ±SD from 3 independent experiments. *P < 0.05; **P < 0.001, Student’s t test.
Resistance to stem falling is an important crop phenotype influenced by many factors ranging from physiology and genetics. Here, we provided evidence that the weaker stem phenotypes of two mutant lines resulted from knockout of a single gene, WRKY13. The forces needed to pull the inflorescence stems apart also decreased significantly in wkry13 mutants. Thus, these mutant lines may
Fig. 6. WRKY13 binds to the NST2 promoter. (A) Schematic of the NST2 promoter. Black bars indicate W-boxes (TGAC). Lines beneath bars indicate the sequences detected by ChIP assays. (B–C) Real-time quantitative PCR of anti-HA ChIP in wrky13-1/PW13:W13 L6 (B) and wild type (C). Stems of 5-week-old transgenic plants were used for ChIP assays. The ACTIN2 promoter (pACTIN2) was used as a negative control. **P < 0.001, Student’s t test. Error bars represent ±SD from three independent ChIP assays. (D) Schematic of the NST2Pro:GUS reporter (bottom) and WRKY13 and GFP effectors (top). Transcription activation of the NST2 promoter reporter by WRKY13 was assessed by GUS activity. GFP was used as a control. Error bars represent ±SD from 3 independent experiments. *P < 0.05, Student’s t test.
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provide good experimental materials to resolve problems caused by yield loss and reduced mechanical harvest efficiency [26]. The most important strength sources to support plants to grow erectly are sclerenchyma cells. To date, how sclerenchyma development is regulated remains largely unknown. In this study, we showed that sclerenchyma cell development was affected in wrky13 mutants. In addition, close examination of inflorescence stem anatomy revealed that stem width, vascular bundle number and the percentage of sclerenchyma area were remarkably reduced in wrky13 mutants (Fig. 3). Examination of the lignin content level displayed that the lignin synthesis was repressed in wrky13 stems (Fig. 4). Wang et al. reported that the disruption of AtWRKY12 initiated the increase of lignin content in the stem [14]. Another article reported that MlWRKY12, an ortholog of AtWRKY12, had similar function of repressing lignin synthesis [27]. Thus WRKY13 is a positive factor while WRKY12 is a negative one for lignin synthesis. Because the best A. thaliana protein match of WKRY12 is WKRY13 (http://www.arabidopsis.org/servlets/TairObject?id=500231630 &type=locus), we hypothesized that plants evolved a pair of regulators from the same ancestor, which guaranteed proper development. These two antagonistic genes may regulate different subregions of stems, a possibility that should be further elucidated. This antagonism may be a mechanism that limits wasteful carbon allocation into stem cells that are not essential to support the plant against gravity. As shown in Fig. 2A and E, WRKY13 was expressed in root. Many studies have reported mutants of altered xylem development in roots of Arabidopsis [28,29]. Since the vascular bundles formation was repressed in wrky13 stems, we wondered whether WRKY13 had a role in the xylem development in roots, which deserves further examinations. Besides lignin-synthesis related enzymes, the sclerenchyma cell development is also regulated by many secondary wall associated factors. We reported here that the level of several NAC genes was reduced in wrky13 mutants (Fig. 5B). Further assays indicated that WRKY13 could directly bind to the promoter of NST2 and activate the NST2 expression (Fig. 6). Previous studies on WRKY12 proved that NST2 was also a downstream target of WRKY12 and its expression was repressed by WRKY12 [14]. We compared the exact recognition sites of WRKYs on FUL promoter. As shown in Supplementary Fig. S4, WRKY13 binds to the 1st, 6th and 7th W-box, while WRKY12 binds to the 2nd W-box. We wondered if the binding of the two WRKYs to NST2 promoter would affect one another. This issue could help us to explain the antagonistic roles of the two WRKYs. Many studies have reported the correlation of sclerenchyma cell reduction and thinner walls [30–32]. However, recently Bao et al. reported that the vln2 vln3 double mutants, with defects in sclerenchyma development, had similar cell-wall thickness of interfascicular fiber cells compared to WT [33]. We have showed that the sclerenchyma development and secondary-wallassociated genes were repressed in wrky13 mutants. We wondered whether the fiber wall thickness of wrky13 mutants was also altered. In fact, the pith cell wall thickness was increased in wrky12 stems. Considering the antagonistic roles of two WRKYs, we cannot exclude the possibility that the mutation of WRKY13 altered the fiber wall thickness.
Author contribution statement W.L., Z.T. and D.Y. conceived and designed research. W.L. conducted experiments, analyzed data, and wrote the article. Z.T. and D.Y. helped to interpret data and edit the article. All authors read and approved the manuscript.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements We thank Dr. Zhixiang Chen (Department of Botany and Plant Physiology, Purdue University, West Lafayette, Indiana, USA) for the wrky13-1, wrky13-2 mutant seeds. We also thank Dr. Zhong Zhao (School of Life Sciences, University of Science and Technology of China, Hefei, China) and Chengbin Xiang (School of Life Sciences, University of Science and Technology of China, Hefei, China) for providing experimental materials and phytotron. This work was supported by the Natural Science Foundation of China (U1202264 Q3 and 31171183) and the Science Foundation of the Chinese Academy of Sciences (KSCX3-EW-N-07 and the CAS 135 program XTBG-F04).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2015. 04.004.
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Contents lists available at ScienceDirect
Plant Science journal homepage: www.elsevier.com/locate/plantsci
WRKY13 acts in stem development in Arabidopsis thaliana
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Wei Li a,b , Zhaoxia Tian a , Diqiu Yu b,∗ a
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School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, Yunnan 666303, China
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Article history: Received 19 October 2014 Received in revised form 23 March 2015 Accepted 3 April 2015 Available online xxx
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Keywords: WRKY13 Weaker stems Sclerenchyma Lignin Arabidopsis thaliana
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1. Introduction
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Stems are important for plants to grow erectly. In stems, sclerenchyma cells must develop secondary cell walls to provide plants with physical support. The secondary cell walls are mainly composed of lignin, xylan and cellulose. Deficiency of overall stem development could cause weakened stems. Here we prove that WRKY13 acts in stem development. The wrky13 mutants take on a weaker stem phenotype. The number of sclerenchyma cells, stem diameter and the number of vascular bundles were reduced in wrky13 mutants. Lignin-synthesis-related genes were repressed in wrky13 mutants. Chromatin immunoprecipitation assays proved that WRKY13 could directly bind to the promoter of NST2. Taken together, we proposed that WRKY13 affected the overall development of stem. Identification of the role of WRKY13 may help to resolve agricultural problems caused by weaker stems. © 2015 Elsevier Ireland Ltd. All rights reserved.
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Despite the organ shape varying in plant species (such as leaves and flowers), many dicotyledonous plants have similar stem structures, which are comprised of epidermis, cortex, vascular bundles, interfascicular tissues, and pith. Unlike parenchyma cells in pith, cells in vascular bundles and interfascicular tissues are sclerenchyma with two kinds of cell walls, primary and secondarily-thickened. Secondary walls are deposited inside the primary walls after cells cease their expansion [1]. The secondary cell walls in plants can be viewed as thermochemical energy storage systems and are considered as an important renewable source of bioenergy [2,3]. Secondary walls also provide mechanical strength to the stem and enable vascular plants to withstand transpirationcaused negative pressure in their vessels [4]. Lignin is an aromatic polymer deposited in secondary thickened cells where it provides strength and impermeability to the
Abbreviations: SND1, secondary wall-associated NAC domain 1; NST1, NAC secondary wall thickening promoting factor1; GUS, -glucuronidase; X-gluc, 5-bromo-4-chloro-3-indolyl b-d-glucuronic acid; ChIP, chromatin immunoprecipitation; PAL4, phenylalanine ammonia-lyase 4; 4CL1, 4-coumarate:coa ligase 1; CesA7, cellulose synthase catalytic subunit 7; CesA8, cellulose synthase 8; CCoAOMT1, caffeoyl coenzyme A O-methyltransferase 1; CAD-c, cinnamyl alcohol dehydrogenase; F5H, ferulate 5-hydroxylase; HCT, hydroxycinnamoyl transferase; CCR1, cinnamoyl CoA reductase 1. ∗ Corresponding author. Tel.: +86 871 5178133; fax: +86 871 5160916. E-mail address: [email protected] (D. Yu).
wall [5]. In dicot plants, the formation of lignin is controlled by many enzymes, such as: phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (4CL) and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) [6]. Besides lignin, the secondary cell wall is composed of many other components. To ensure proper growth, biosynthesis of these cell wall components is highly coordinated via a cascade of transcription factors. Several transcription factors, including the NAC and MYB families, have been implicated in regulating cell wall biosynthesis or deposition. Two NAC genes, NAC secondary wall thickening promoting factor1 (NST1) and NST2, act redundantly in secondary wall thickening in the endothecium [7]. Disruption of MYB26 can cause failure of secondary wall thickening in anther endothecium [8]. In Arabidopsis thaliana, the WRKY transcription factor superfamily has 74 members with diverse functions [9,10]. For example, WRKY57 functions as a node of convergence for JA- and auxinmediated signals in leaf senescence [11]. wrky8 mutant plants are even more hypersensitive to salt and susceptible to cruciferinfecting tobacco mosaic virus infection [12,13]. Disruption of WRKY12 initiates pith secondary wall formation and increases stem biomass in dicotyledonous plants [14]. In this report, we provide evidence that mutation of WRKY13 leads to a weaker stem phenotype in A. thaliana, and WRKY13 is a positive regulator for overall stem development. Stem diameter and the number of vascular bundles are significantly decreased in wkry13 mutants. WRKY13 upregulates transcript levels of lignin pathway genes and lignin content in stem. In addition, the secondary cell wall synthesis gene NST2 is proved to be the direct downstream target of WRKY13.
http://dx.doi.org/10.1016/j.plantsci.2015.04.004 0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.
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2. Materials and methods
2.6. ChIP assays
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2.1. Plant growth conditions
Chromatin immunoprecipitation (ChIP) assays were performed essentially as previously described [17,18]. Inflorescence stems of wild type (WT) and wrky13-1/PW13:W13 L6 transgenic plants were used for ChIP assays. HA antibody (Thermo Fisher Pierce, Rockford, IL, USA) was used to immunoprecipitate protein–DNA complex, and the precipitated DNA was purified by using a PCR purification kit for real-time quantitative-PCR analysis. The ACTIN7 (AT5G09810) 5 untranslated region sequence was used as an endogenous control. The relative quantity value is presented as the DNA binding ratio (differential site occupancy). The primers used for different promoters are listed in Supplementary Table S1.
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A. thaliana (accession Columbia) seeds (obtained from the Arabidopsis Information Resource, http://www.arabidopsis.org) were surface sterilized with 20% bleach and washed three times with sterile water. Sterilized seeds were vernalized in darkness for 2 days at 4 ◦ C, planted in soil, and then grown in a tissue culture room at 22 ◦ C and 75% humidity under a 16 h-light/8 h-dark photoperiod. 2.2. Expression analysis For real-time RT-PCR analysis, total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I (Fermentas, Vilnius, Lithuania) according to the manufacturer’s instructions. Total RNA (∼2 g) was reversetranscribed in a 20-L reaction mixture by using Superscript II (Invitrogen). After that, 1-L aliquots were used as templates for real-time RT-PCR. Reactions (20 L) were performed with a Lightcycler FastStart DNA Master SYBR Green I Kit (Roche, Penzberg, Germany) in a Roche LightCycler 480 real-time PCR machine, according to the manufacturer’s instructions. ACTIN2 (AT3G18780) was used as a control, and three biological replicates were conducted. The primers used for real-time RT-PCR amplification of different genes are listed in Supplementary Table S1. For -glucuronidase (GUS) staining, histochemical detection of GUS activity was performed with 5-bromo-4-chloro-3-indolyl b-d-glucuronic acid (X-gluc) as the substrate. Plant tissues were prefixed in ice-cold 90% (vol/vol) acetone for 20 min, and then washed three times with GUS staining buffer (without X-gluc) before incubation in X-gluc solution [1 mM X-gluc, 50 mM NaPO4 (pH 7), 1 mM K3 Fe(CN)6 , 1 mM K4 Fe(CN)6 , and 0.05% Triton X-100] under a vacuum for 10 min at room temperature and then incubated overnight at 37 ◦ C. Several changes of 70% (vol/vol) ethanol were used to remove the chlorophyll. 2.3. Measurement of breaking force The basal parts of 6-week-old inflorescence stems were measured. The force needed to pull samples apart was measured through dynamic mechanical analysis with a DMA Q800 (TA Instruments, New Castle, DE, USA). Ends of each stem segment were clamped at the same distance and torn apart at the same speed. 2.4. Tissue sections and microscopy analysis
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Stem samples were fixed in FAA buffer (formaldehyde:glacial acetic acid:50% ethanol, 1:1:18), and then embedded in paraffin. Specimens were cut into 8-m-thick sections and stained with 0.05% toluidine blue O and observed under Zeiss Axio Scope (Zeiss, Oberkochen, Germany).
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2.5. Lignin measurements
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2.7. GUS activity assays Constructs were introduced into Agrobacterium tumefaciens (strain GV3101), and infiltration of Nicotiana benthamiana was performed as described previously [12]. Infected tissues were analyzed for 48 h after infiltration. GUS activity was measured as previously described [19] except for extracts which were not first purified by column chromatography.
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Thioglycolic acid (TGA) assay was carried out according to Campbell and Ellis [15] and Jones et al. [16]. The crude cell-wall preparations were saponified in 1 M NaOH before being extracted through TGA for 3 h at 80 ◦ C. The insoluble material was collected by centrifugation, washed with distilled water, incubated in 1 M NaOH overnight and set on a rotating shaker at room temperature. The supernatant was transferred with 200 mL concentrated HCl added. The precipitate was collected and resuspended in 1 M NaOH. The OD280 was read under a tenfold sample dilution.
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3. Results
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3.1. The wrky13 mutants take on a weaker stem phenotype
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We screened several transfer (T)-DNA insertion mutants obtained from the Arabidopsis Biological Resource Center for mutants of the stem phenotype. Two T-DNA insertion mutants, wrky13-1 (SALK 064346C) and wrky13-2 (SALK 032912) [20], were isolated. These two lines were predicted to have T-DNA insertions in the AtWRKY13 (AT4G39410) gene (Fig. 1A). Then, RNA of the two mutant plants was extracted and subjected to RT-PCR. No full-length transcript was detected in either wkry13-1 or wrky13-2 (Fig. 1B). Under long-day conditions, mutation in WRKY13 did not lead to obvious developmental problem during the seedling stage (Fig. 1C). However, in mature plants, wrky13-1 and wrky13-2 displayed a weaker stem phenotype, while other organs grew normally (Fig. 1C). To ascertain the function of WRKY13 in stems, we fused HA tag and WRKY13 CDS into the same expression vector driven by the WRKY13 promoter (Supplementary Fig. S1A). The construct was transformed into wrky13-1 to generate wrky13-1/PW13:W13. Transcripts of WRKY13 were detected in transgenic plants (Supplementary Fig. S1B). WRKY13 was sufficient to rescue the weaker stem phenotype of wkry13-1 (Supplementary Fig. S1C). We wondered if this weaker stem phenotype of wrky13-1 indicated that the mechanical strength necessary to support the plant was lower in mutant plants. As shown in Fig. 1D, the forces needed to pull wrky13-1 mutant stems apart decreased sharply, while the breaking force for the wrky13-1/PW13:W13 transgenic stems was similar to what required for WT. 3.2. Expression analysis of WRKY13
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Since wrky13 mutant stems developed weaker stem phenotype, we investigated the expression pattern of WRKY13, especially in stems. RNA extracted from different organs of WT plants was used to perform real-time RT-PCR. We found that WRKY13 was preferentially expressed in stem internodes, where its transcript level increased with maturity (Fig. 2A). WRKY13 gene transcripts were also detected in roots. To further elucidate the expression pattern of WRKY13, we fused a 2.5 kb promoter of WRKY13 to a -glucuronidase (GUS) reporter. The construct was used to transform WT Arabidopsis. Several lines
Please cite this article in press as: W. Li, et al., WRKY13 acts in stem development in Arabidopsis thaliana, Plant Sci. (2015), http://dx.doi.org/10.1016/j.plantsci.2015.04.004
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Fig. 1. wrky13 mutants developed weaker stem phenotype. (A) Characterization of WRKY13 (AT4G39410) gene. The exons, introns and untranslated regions are represented by black boxes, black lines and white boxes, respectively. The T-DNA insertion lines are designated wrky13-1 (Salk 064346C) and wrky13-2 (Salk 032912) with insertions in exons. A pair of primers (W13F and W13R) are indicated as arrows. (B) Aaerial part of 5-week-old plants were harvested for RNA extraction. Primers W13F and W13R were used to determine WRKY13 transcript levels. ACTIN2 was used as an internal control. (C) wrky13 mutants developed weaker stems compared to that of WT. Plants were grown under long-day conditions. Scale bars represent 1 cm. (D) Less force is needed to break wrky13 mutant inflorescence stems. The basal part of inflorescence stem of 6-week-old plants were analyzed. Error bars represent ±SD from 15 independent experiments. **P < 0.001, Student’s t test.
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Fig. 2. Expression pattern of WRKY13. (A) Long-day grown WT plants were used for RNA extraction. Quantitative RT-PCR showing expression of WRKY13 in different organs normalized to expression of ACTIN2. Error bars represent ±SD from 3 independent experiments. *P < 0.05; **P < 0.001, Student’s t test. (B–E) Expression patterns determined by the WKRY13 promoter--glucuronidase (GUS) construct. At least 5 independent transgenic lines showed similar expression pattern. (F) Cross section of WKRY13 promoterGUS transgenic plant stem. The section is 8 m thick. GUS activity is detected in secondary wall-containing cell types. (G) High magnification showing the detailed expression in stem sections. Scale bars in (F) and (G) represent 50 m.
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Fig. 3. The development of sclerenchyma cells was repressed in wrky13 mutants. (A and B) Cross-sections of the basal part of 6-week-old inflorescence stems of wild type (A) and wrky13-1 (B) were subjected to toluidine blue O staining. (C and D) Enlarged portion of A and B, respectively. ph, phloem; xy, xylem; if, interfascicular fibers. Scale bars represent 100 m. (E) Schematic representation of methods used in (H)–(J). The red circle indicates cross sectional area of entire stem. The area within two yellow circles represents sclerenchyma cell area. (F–J) Quantitative statistics of stem cross-sections. Error bars represent ±SD from 8 independent experiments. *P < 0.05; **P < 0.001, Student’s t test.
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of transgenic plants showed similar results and confirmed the preferential expression in inflorescence stems (Fig. 2B). GUS signals were also detected in leaf vein, root and base of pod (Fig. 2C–E). In addition, we cut thin cross-sections of GUS transgenic plants to investigate the expression pattern of WRKY13 in stem. As shown in Fig. 2F and G, GUS activities were detected in all the cells in the stele. This indicates that WRKY13 may function in stem development. 3.3. The development of sclerenchyma is repressed in wrky13 mutants To investigate the anatomical defects of mutants, thin crosssections of stems were cut and subjected to toluidine blue O staining. As shown in Fig. 3, the stem diameter decreased significantly from 890 ± 25 m for WT (Fig. 3A) to 676 ± 19 m and 674 ± 20 m for wrky13-1 (Fig. 3B) and wrky13-2 (Supplementary Fig. S2A and Fig. 3F), respectively. The stem diameter of wrky131/PW13:W13 L6 (Supplementary Fig. S2B) is 894 ± 27 m, which was similar to that of WT (Fig. 3F). The number of vascular bundles decreased from 6.3 ± 0.4 for WT to 4.2 ± 0.2 and 4.6 ± 0.3 for wrky13-1 and wrky13-2 plants, respectively (Fig. 3G). There was no significant difference in the number of vascular bundles between WT and wrky13-1/PW13:W13 L6 (Fig. 3G). In addition, we measured cross-sectional area of the entire inflorescence stem and cross-sectional area of the area containing sclerenchyma cells (Fig. 3E). The cross-sectional area of the entire stem decreased from 128 ± 6.1 and 123 ± 6.4 inch2 for WT and wrky13-1/PW13:W13 L6 to 74 ± 5.5 and 74 ± 5.8 inch2 for wrky13-1 and wrky13-2, respectively (Fig. 3H). The cross-sectional area containing sclerenchyma cells decreased from 50 ± 2.1 and 46 ± 1.8 inch2 for WT and wrky131/PW13:W13 L6 to 21 ± 1.8 and 23 ± 1.7 inch2 for wrky13-1 and wrky13-2, respectively (Fig. 3I). Because mechanical support for the plant is derived mainly from sclerenchyma cells, the ratio of
sclerenchyma cell area divided by cross-sectional stem area could reflect stem mechanical strength. Again, these ratios decreased from 39 ± 1.4 and 37 ± 1.3% for WT and wrky13-1/PW13:W13 L6 to 28 ± 1.1 and 31 ± 1.2% for wrky13-1 and wrky13-2, respectively (Fig. 3J). The defect in sclerenchyma development was further confirmed by staining for lignin with phloroglucinol HCl (Fig. 4A–C). To confirm the reduction in the lignin content of wrky13 stems, thioglycolic acid assays were carried out for stem material. Fig. 4D demonstrates that the lignin content decreased in wrky13 stems. These observations confirmed the sclerenchyma development defects in wrky13 mutants. This conclusion is consistent with the function of sclerenchyma in providing physical support for plants to grow erectly.
3.4. Mutation of WRKY13 alters the expression level of several genes Since the lignin content decreased in wrky13 stems. We wondered if levels of lignin pathway genes were affected by the mutation of WKRY13. Real-time RT-PCR analysis was used to examine gene transcript levels. As shown in Fig. 5A, levels of PAL4 (AT3G10340), 4CL1 (AT1G51680), HCT (AT5G48930), and CAD6 (AT4G37970) were reduced, while CCoAOMT1 (AT4G34050), CCoAOMT7 (AT4G26220), CAD-c (AT3G19450), F5H (AT4G36220), and CCR1 (AT1G15950) remained unchanged in wrky13 stems. We also examined the levels of transcription factors that are related to sclerenchyma secondary wall synthesis [21–24]. The results showed that SND1(AT1G32770), NST1(AT2G46770), NST2 (AT3G61910), CesA7 (AT5G17420), and CesA8 (AT4G18780) were repressed by the disruption of WRKY13. Other genes, such as, C3H14 (AT1G66810) and C3H14L (AT1G68200), were not significantly changed in wrky13 mutants.
Fig. 4. The biosynthesis of lignin was affected in wrky13 mutants. (A-C) Free-hand sections of 6-week-old inflorescence stems were subjected to phloroglucinol-HCl staining. Red area represent lignin. (A) Wild type; (B) wrky13-1; (C) wrky13-2. Scale bars represent 100 m. (D) Thioglycolic acid (TGA) assays were carried out on stem material of WT and mutants. 6-week-old inflorescence stems were used for the TGA assays. Error bars represent ±SD from 8 independent experiments. *P < 0.05, Student’s t test.
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3.5. WRKY13 binds to the NST2 promoter Previous reports indicated that WRKY transcription factors specifically bind to W-box elements containing a TGAC core sequence in their target gene promoters [25]. To investigate the relationship between WRKY13 and its downstream targets, we searched promoters of several genes for W-box and performed in vivo chromatin immunoprecipitation (ChIP) assays by using wkry13-1/PW13:W13 L6 plants. Detailed ChIP analysis indicated that WRKY13 bound to the promoter regions of NST2 (Fig. 6A–C). We did not observe any specific binding of WRKY13 to the tested PAL4, 4CL1, CESA7 and CESA8 promoters (Supplementary Fig. S3). To further confirm the positive regulatory function of WRKY13, we performed transient expression assays. A reporter plasmid was constructed by fusing a 2-kb promoter sequence of NST2 with the -glucuronidase (GUS) reporter gene. The effector plasmid had a WRKY13 or GFP gene driven by the cauliflower mosaic virus (CaMV) 35S promoter. As shown in Fig. 6D, coexpression of the WRKY13 gene produced significantly higher GUS activity than coexpression of the GFP gene. This supported the hypothesis that WRKY13 could directly activate the expression of NST2.
4. Discussion
Fig. 5. Lignin pathway genes and secondary-wall-related genes are altered in wrky13 mutants. (A and B) Stems of 5-week-old WT plants were harvested for RNA extraction. Quantitative RT-PCR was used to analysis genes expression levels. Error bars represent ±SD from 3 independent experiments. *P < 0.05; **P < 0.001, Student’s t test.
Resistance to stem falling is an important crop phenotype influenced by many factors ranging from physiology and genetics. Here, we provided evidence that the weaker stem phenotypes of two mutant lines resulted from knockout of a single gene, WRKY13. The forces needed to pull the inflorescence stems apart also decreased significantly in wkry13 mutants. Thus, these mutant lines may
Fig. 6. WRKY13 binds to the NST2 promoter. (A) Schematic of the NST2 promoter. Black bars indicate W-boxes (TGAC). Lines beneath bars indicate the sequences detected by ChIP assays. (B–C) Real-time quantitative PCR of anti-HA ChIP in wrky13-1/PW13:W13 L6 (B) and wild type (C). Stems of 5-week-old transgenic plants were used for ChIP assays. The ACTIN2 promoter (pACTIN2) was used as a negative control. **P < 0.001, Student’s t test. Error bars represent ±SD from three independent ChIP assays. (D) Schematic of the NST2Pro:GUS reporter (bottom) and WRKY13 and GFP effectors (top). Transcription activation of the NST2 promoter reporter by WRKY13 was assessed by GUS activity. GFP was used as a control. Error bars represent ±SD from 3 independent experiments. *P < 0.05, Student’s t test.
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provide good experimental materials to resolve problems caused by yield loss and reduced mechanical harvest efficiency [26]. The most important strength sources to support plants to grow erectly are sclerenchyma cells. To date, how sclerenchyma development is regulated remains largely unknown. In this study, we showed that sclerenchyma cell development was affected in wrky13 mutants. In addition, close examination of inflorescence stem anatomy revealed that stem width, vascular bundle number and the percentage of sclerenchyma area were remarkably reduced in wrky13 mutants (Fig. 3). Examination of the lignin content level displayed that the lignin synthesis was repressed in wrky13 stems (Fig. 4). Wang et al. reported that the disruption of AtWRKY12 initiated the increase of lignin content in the stem [14]. Another article reported that MlWRKY12, an ortholog of AtWRKY12, had similar function of repressing lignin synthesis [27]. Thus WRKY13 is a positive factor while WRKY12 is a negative one for lignin synthesis. Because the best A. thaliana protein match of WKRY12 is WKRY13 (http://www.arabidopsis.org/servlets/TairObject?id=500231630 &type=locus), we hypothesized that plants evolved a pair of regulators from the same ancestor, which guaranteed proper development. These two antagonistic genes may regulate different subregions of stems, a possibility that should be further elucidated. This antagonism may be a mechanism that limits wasteful carbon allocation into stem cells that are not essential to support the plant against gravity. As shown in Fig. 2A and E, WRKY13 was expressed in root. Many studies have reported mutants of altered xylem development in roots of Arabidopsis [28,29]. Since the vascular bundles formation was repressed in wrky13 stems, we wondered whether WRKY13 had a role in the xylem development in roots, which deserves further examinations. Besides lignin-synthesis related enzymes, the sclerenchyma cell development is also regulated by many secondary wall associated factors. We reported here that the level of several NAC genes was reduced in wrky13 mutants (Fig. 5B). Further assays indicated that WRKY13 could directly bind to the promoter of NST2 and activate the NST2 expression (Fig. 6). Previous studies on WRKY12 proved that NST2 was also a downstream target of WRKY12 and its expression was repressed by WRKY12 [14]. We compared the exact recognition sites of WRKYs on FUL promoter. As shown in Supplementary Fig. S4, WRKY13 binds to the 1st, 6th and 7th W-box, while WRKY12 binds to the 2nd W-box. We wondered if the binding of the two WRKYs to NST2 promoter would affect one another. This issue could help us to explain the antagonistic roles of the two WRKYs. Many studies have reported the correlation of sclerenchyma cell reduction and thinner walls [30–32]. However, recently Bao et al. reported that the vln2 vln3 double mutants, with defects in sclerenchyma development, had similar cell-wall thickness of interfascicular fiber cells compared to WT [33]. We have showed that the sclerenchyma development and secondary-wallassociated genes were repressed in wrky13 mutants. We wondered whether the fiber wall thickness of wrky13 mutants was also altered. In fact, the pith cell wall thickness was increased in wrky12 stems. Considering the antagonistic roles of two WRKYs, we cannot exclude the possibility that the mutation of WRKY13 altered the fiber wall thickness.
Author contribution statement W.L., Z.T. and D.Y. conceived and designed research. W.L. conducted experiments, analyzed data, and wrote the article. Z.T. and D.Y. helped to interpret data and edit the article. All authors read and approved the manuscript.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements We thank Dr. Zhixiang Chen (Department of Botany and Plant Physiology, Purdue University, West Lafayette, Indiana, USA) for the wrky13-1, wrky13-2 mutant seeds. We also thank Dr. Zhong Zhao (School of Life Sciences, University of Science and Technology of China, Hefei, China) and Chengbin Xiang (School of Life Sciences, University of Science and Technology of China, Hefei, China) for providing experimental materials and phytotron. This work was supported by the Natural Science Foundation of China (U1202264 Q3 and 31171183) and the Science Foundation of the Chinese Academy of Sciences (KSCX3-EW-N-07 and the CAS 135 program XTBG-F04).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2015. 04.004.
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