An Arabidopsis WDR protein coordinates cellular networks involved in light, stress response and hormone signals

Plant Science 241 (2015) 23–31

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An Arabidopsis WDR protein coordinates cellular networks involved in light, stress response and hormone signals Huey-wen Chuang ∗ , Ji-Huan Feng, Yung-Lin Feng, Miam-Ju Wei Department of Bioagricultural Sciences, National Chiayi University, Chiayi, Taiwan

a r t i c l e

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Article history: Received 14 August 2015 Received in revised form 25 September 2015 Accepted 25 September 2015 Available online 30 September 2015 Keywords: WD-40 protein Biotic stress Abiotic stress Light signaling Hormone signaling

a b s t r a c t The WD-40 repeat (WDR) protein acts as a scaffold for protein interactions in various cellular events. An Arabidopsis WDR protein exhibited sequence similarity with human WDR26, a scaffolding protein implicated in H2 O2 -induced cell death in neural cells. The AtWDR26 transcript was induced by auxin, abscisic acid (ABA), ethylene (ET), osmostic stress and salinity. The expression of AtWDR26 was regulated by light, and seed germination of the AtWDR26 overexpression (OE) and seedling growth of the T-DNA knock-out (KO) exhibited altered sensitivity to light. Root growth of the OE seedlings increased tolerance to ZnSO4 and NaCl stresses and were hypersensitive to inhibition of osmotic stress. Seedlings of OE and KO altered sensitivities to multiple hormones. Transcriptome analysis of the transgenic plants overexpressing AtWDR26 showed that genes involved in the chloroplast-related metabolism constituted the largest group of the up-regulated genes. AtWDR26 overexpression up-regulated a large number of genes related to defense cellular events including biotic and abiotic stress response. Furthermore, several members of genes functioning in the regulation of Zn homeostasis, and hormone synthesis and perception of auxin and JA were strongly up-regulated in the transgenic plants. Our data provide physiological and transcriptional evidence for AtWDR26 role in hormone, light and abiotic stress cellular events. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The WD (Trp-Asp)-40 repeat (WDR) was first discovered in the ␤-subunit of heterotrimeric GTP-binding proteins (G proteins) [1]. G proteins are implicated in diverse cellular events through G protein-coupled receptor (GPCR) signaling to downstream targets, which may ultimately result in transcriptional regulation [2]. The WD-40 repeat motif consists of repeating amino acid sequences separated by approximately 40 residues and contains amino acids, tryptophan and aspartate (WD) at the end of the repeat. WDR proteins commonly contain four to eight WD-40 repeats; however, the separating distance and actual amino acid sequence within individual repeats is highly variable. The WD repeat domains form a platform for the assembly of multiple proteins and therefore play key roles in the formation of protein–protein complexes [3]. It is now believed that the WD-40 motifs may contribute to the formation of a stable protein complex that participates in various signaling pathways. In the human genome, the function of the WDR

∗ Corresponding author. Fax: +886 5 271 7755. E-mail address: [email protected] (H.-w. Chuang). http://dx.doi.org/10.1016/j.plantsci.2015.09.024 0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.

protein shows a high degree of diversity, including signal transduction, RNA processing, cell cycle control, transcriptional regulation, vesicular trafficking, regulation of cytoskeleton assembly, nuclear export, RNA processing, and chromatin modification [4]. In the Arabidopsis genome, more than 200 putative WD-40 domain-containing proteins have been predicted [5]. A number of the WDR proteins have been functionally characterized to play a role in light signaling, photomorphogenesis, and flowering, such as Constitutively photomorphogenic 1 (COP1), Suppressor of Phy-A-105 (SPA1), and Anthocyanin11 (AN11) [6,7]. Several WDR proteins play a role in gametogenesis [8], abiotic stress [9], auxinregulated embryogenesis [10], meristem maintenance [11], floral development [12], and metal ion binding [13]. Understanding of the molecular mechanism indicates that WDR proteins may alter the cellular homeostasis of reactive oxygen species (ROS) through protein interactions with components such as histones, ribosome biogenesis proteins, or chromatin assembly factors to participate in developmental regulation [9,11,14–16]. ROS molecules are crucial for plant development, and an increased ROS level is necessary for plants to initiate proper signals for acclimation responses to environmental stress [17]. For example, light-induced auxin biosynthesis occurs through the control of ROS production [18].

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Fig. 1. Homologous genes of AtWDR26. Sequence alignment between AtWDR26 and human WDR26 (A). Letters in black and grey shadow indicate the identical and conserved amino acid residues, respectively. The deduced amino acid sequence of AtWDR26 contained the LisH, CTLH, and WD (WD-40) domains (B). Homologous genes of ATWDR26 identified in diverse plant species. a indicates the amino acid (a.a) residues of the coding region, b indicates % of positive a.a residues, c indicates % of identical a.a residues (C).

Fig. 2. Expression of AtWDR26. RT-PCR using primers specific to the coding sequence of AtWDR26 was conducted to detect the AtWDR26 expression. RNA isolated from different parts of tissues of 4-week-old Arabidopsis plants was used for RT-PCR analysis for detecting tissue specific expression of AtWDR26 (A). RNAs isolated from 7-day-old whole seedlings treated with different hormones including 10 ␮M IAA, 150 ␮M ABA and 50 ␮M ACC, and abiotic stresses including 150 mM NaCl and 300 mM mannitol (man) were used for RT-PCR analysis to detect the AtWDR26 inducible expression (B). RNAs isolated from the 7-day-old whole seedlings grown under the light (100 ␮mol s−1 m−2 ) and dark conditions at 23 ◦ C were used for RT-PCR analysis to demonstrate the light inducible expression (C). Actin 2 (ACT2) expression was used as an internal control. The gene expression levels of silique in (A), water in (B) and dark in (C) were used as the bases for comparative expression.

Ethylene (ET), jasmonic acid (JA) and ABA function in the control of stomatal closure through the modulation of ROS levels in the guard cells [19–21]. ET and salicylic acid (SA) are implicated in the crosstalk between high light acclimation and disease resistance through mediation of ROS molecules [22,23]. Plant acclimation to high light stress results in reduced stomatal conductance, increased temperature, and increased activity of heat shock transcription factors [24]. In addition, higher levels of ABA and auxin accumulate in plant tissues subjected to high light stress [25,26]. Human WDR26, a novel member of the WD-40 gene family, functions in the regulation of H2 O2 homeostasis, cell movement, and apoptosis [27]. Arabidopsis homolog of human WDR26, designated as AtWDR26 (At5G08560), has been identified as an

interacting protein of the Arabidopsis RanBPM (Ran-binding protein in the microtubule-organizing center), a component of the C-terminal to the LisH motif (CTLH) complexes [28]. In yeast, the CTLH complex of Gid/Vid proteins plays a role in vacuole and proteasome-dependent fructose-1,6-bisphosphatase degradation [29]. The mammalian CTLH complexes also exhibit a similar function in lysosome and proteasome-dependent proteolysis [30]. The AtRanBPM complex has been identified as a cytosolic protein complex; however, its cellular function remains unknown [28]. The function of AtWDR26 was determined in the current study. The physiological and transcriptional evidence suggests that AtWDR26 is a component implicated in the crosstalk regulation between light, hormone and abiotic stress response.

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Fig. 3. Verification of light-related phenotypes of AtWDR26OE and KO lines. Seed germination rates of wild-type and 2 independent transgenic lines, OE-1 and OE-5, were calculated 5-day after sowing under light-grown condition (n = 50) (A). Seedlings of wild-type and 2 KO lines, T-1 and T-2, were germinated under fluorescence white light. The cotyledons of KO lines exhibited darker green cotyledons after 4-day-culture, but similar to wild-type hypocotyl growth (n = 50) (B). Presented data are means ± S.E. from three independent experiments.

Fig. 4. Function of AtWDR26 in abiotic stress response. The 4-day-old seedlings of wild-type and transgenic plants, OE-1 and OE-5, were transferred onto media containing 300 ␮M ZnSO4 (A), 150 mM NaCl (B), and 300 mM mannitol (C). The root growth was measured 4 days after subculture (n = 20). The relative root growth was calculated by dividing the root growth from treated plants with that of cultured in 1/2 MS medium. Seed germination rates of KO lines, T-1 and T-2, on 1/2 MS media containing 150 mM NaCl (D) and 300 mM mannitol (E) were calculated 5 days after sowing (n = 50). Relative seed germination rates were calculated by dividing seed germination rates of treated plants with those of control. Each experiment was performed in triplicate. Presented data are means ± S.E. from three independent experiments.

2. Materials and methods 2.1. Plant materials and growth conditions All Arabidopsis lines used in this study are in the Columbia background. T-DNA knock-out (KO) lines including SALK 095495

(T-1) and SALK 100917 (T-2) were obtained from the Arabidopsis Biological Resource Center (ABRC). Seeds sterilized with 3% sodium hypochlorite solution were incubated at 4 ◦ C for 3 days before germination assays. A seed germination test was performed by sowing sterilized seeds on 1/2 MS medium [31] containing 150 mM NaCl and 300 mM mannitol. The seed germination rate

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Fig. 5. Function of AtWDR26 involved in hormone signaling transduction. The 4-day-old seedlings of wild-type and OE plants, OE-1 and OE-5, were transferred onto media containing 2,4-D (10, 30, and 50 nM) (A), 50 ␮M of MeJA (B), and 50 ␮M ABA (C). Root length measured 4 day after culture was used for calculation of relative root growth (n = 20). Seeds of OE-1 and OE-5 were germinated on medium containing 5 ␮M of ACC under dark condition. The hypocotyl growth root growth was measured 4 days after sowing and used for calculation of relative hypocotyl growth (n = 50) (D).

was scored 5 days after sowing. The relative seed germination rate was calculated by dividing the germination rate of treated seeds by the germination rate of control seeds. A root growth assay was conducted by transferring 4-day-old seedlings to medium containing various concentration of 2,4-D (10, 30 and 50 nM), 50 ␮M of methyl–jasmonic acid (MeJA), and 50 ␮M ABA. Root growth was measured 4 days after transferring. The relative root growth was calculated by dividing the root length of treated plants by that of the control. All tested plants were cultured under long-day lighting conditions. Hypocotyl growth was determined by germinating sterilized seeds on medium containing 5 ␮M 1aminocyclopropanecarboxylic acid (ACC) under dark conditions. The hypocotyl length of each seedling was measured 4 days after sowing. All plants were grown at 23 ◦ C. Each experiment was performed in triplicate. Presented data are means ± S.E. from three independent experiments. 2.2. Overexpression of AtWDR26 cDNA in Arabidopsis The full length cDNA fragment of AtWDR26 was generated by RT-PCR using primers specific to the 5 and 3 ends of the cDNA sequence of the Arabidopsis gene At5G08560. The obtained cDNA was digested and ligated into the NcoI/SpeI sites of pCambia 1302 (CAMBIA). This 35S-AtWDR26 construct was transferred into Agrobacterium strain GV3101 and used to transform

Arabidopsis following the floral dip method [32]. Original transgenic seeds (T0) were selected on MS medium containing 15 ␮g/mL hygromycin. Following two rounds of selection, T3 homozygous lines were generated and used for physiological analysis.

2.3. Verification of T-DNA insertion lines To isolate homozygous KO mutants, leaf tissues from soilgrown Arabidopsis were used for genomic DNA extraction based on the Quick DNA Prep method for PCR described in Weigel and Glazebrook [33]. To genotype the plants with the T-DNA insert, PCR was performed on genomic DNA using primers specific to the T-DNA left border (LBa1, 5 -TGGTTCACGTAGTGGGCCATCG-3 ) and AtWDR26 sequence (AWDR-F 5 -ATGGGAGTTGTGGAGGATAC3 ). The wild-type genomic sequence of the AtWDR26 gene was amplified using primers specific to the 5 -end (AWDR-F 5 -ATGGGAGTTGTGGAGGATAC-3 ) and the 3 -end (AWDR-R 5 ATTCCCATTGCATCGGTGAA-3 ) of the coding sequence. The PCR amplification was carried out for 35 cycles under the following conditions: 95 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 3 min. To examine gene knock-out of AtWDR26 in the T-DNA insertion lines, total RNA isolated from homozygous T-DNA lines was used for RT-PCR analysis using the AtWDR-1-F and AtWDR-1-R primers specific to the 5 - and 3 -end, respectively, of the coding region of AtWDR26.

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Fig. 6. Transcriptome alterations by AtWDR26 overexpressing. Gene groups up-regulated in transgenic plants overexpressing AtWDR26 (A). Subclasses of genes in the gene group of metabolism (B). Subclasses of genes in the gene group of stress-related (C) and transcription factors (D).

2.4. RNA extraction and RT-PCR analysis For detecting tissue specific expression, different tissues harvested from 4-week-old mature wild-type Arabidopsis were used for RNA extraction. For the examination of gene expression in response to various environmental cues, 7-day-old seedlings cultured in 1/2 MS liquid medium were treated with various reagents including 10 ␮M IAA, 150 ␮M ABA, 50 ␮M ACC, 150 mM NaCl, and 300 mM mannitol for 24 h and used for RNA extraction. For investigating light inducible expression, 7-day-old seedlings grown in the 1/2 MS medium under 24 h light and dark conditions were harvested for RNA extraction. Total RNA extraction for Arabidopsis tissues followed the method described by Parcy et al. [34]. For RTPCR, 1 ␮g of total RNAs was used for the first-strand cDNA synthesis using ImProm-II Reverse transcriptase (Promega). The synthesized cDNA was subsequently used for PCR amplification using primers specific to gene coding sequences of AtWDR26. The actin2 gene expression was used as an internal control. The PCR amplification for RT-PCR analysis was carried out under the following conditions: 95 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 1 min; 24 cycles for actin 2 and 26–34 cycles for the remainder of the tested genes. PCR products were separated on 0.8% agarose gel. The gel image was quantified by Quantity One software (Bio-Rad). The PCR DNAs amplified by gene specific primers were normalized by actin 2 DNA. The relative fold changes were calculated by dividing the signals of treatments with that of control. 2.5. Microarray analysis Total RNA (1 ␮g) isolated from seven-day-old wild-type seedlings of the AtWDR26 overexpression line was labeled with

Cy3-CTP or Cy5-CTP and used to hybridize the Agilent Whole Arabidopsis Genome 4 × 44k oligo microarray. Fluorescent probe preparation, hybridization, washing, and scanning were performed according to the manufacturer’s protocols. 3. Results 3.1. Sequence and expression analysis of AtWDR26 AtWDR26 is a member of the human WDR26 (AY304473) WD-40 gene family [35]. A database search using the BLASTP algorithm indicated that the predicted amino acid sequence of AtWDR26 exhibited 54% similarity with human WDR26 (Fig. 1A). Human WDR26 is a scaffolding protein that interacts with components of heterotrimeric G proteins [36] and is a member of the CUL4- and DDB1-associated WDR protein (CDW) family [37]. Functional characterization showed that WDR26 expression suppresses the transcription activity of the MAPK signaling pathway [35]. The protein domains of AtWDR26 were identified using the Simple Modular Architecture Research Tool (SMART) [38]. Sequence analysis indicated that AtWDR26 contained one of the Lis homology domains (LisH), the C-terminal to the LisH motif (CTLH) [39], and 7 WD-40 repeats (Fig. 1B). Sequence analysis using BLAST identified 10 genes from different plant species (including Arabidopsis) that exhibited >60% similarity with the AtWDR26 sequence (Fig. 1C). A homologous sequence was also found in Rhizophagus irregularis, in which AtWDR26 shared 57% similarity with a gene coding for a subunit of the glucose-induced degradation (GID7) complex (Fig. 1C). In Arabidopsis, AtWDR26 is identified as an interacting protein of the Arabidopsis Ran-binding protein in the microtubule-organizing center (RanBPM) complex that contains

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highly conserved SPRY, LisH, CTLH and CRA domains [28]. However, the signaling pathway regulated by AtWDR26 remains uncharacterized. To predict the biological function, the tissue specific expression of AtWDR26 was performed. Our results showed that the AtWDR26 transcript was expressed in the flowers, leaves and roots, but not in the siliques (Fig. 2A). Moreover, the AtWDR26 transcript was induced by hormones including IAA, ABA, and ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and osmotic stress including 300 mM mannitol and 150 mM NaCl (Fig. 2B). Accumulated data suggest that WD-40 repeat proteins function as scaffold proteins in protein interactions in diverse cellular events including abiotic stress and light signaling [6,40]. To analyze the function of AtWDR26 in the light-related physiological response, light-regulated expression of AtWDR26 was confirmed using RTPCR, which showed that the AtWDR26 transcript was expressed in the light-grown seedlings and absent in the dark-grown seedlings (Fig. 2C). 3.2. AtWDR26 expression altered seed germination and seedling growth To investigate the biological function of AtWDR26, the full length cDNA of AtWDR26 was constitutively expressed in Arabidopsis. Four homozygous transgenic lines were obtained. Transgene insertion in the transgenic lines was confirmed by the Southern blot analysis (supplemental 1A). Two transgenic plants, OE-1 and OE-5 with overexpression of AtWDR26 were selected for physiological analysis (supplemental 1B). Moreover, 2 T-DNA knockout (KO) lines with insertions in the first exon of AtWDR26 genomic sequence were used in this study (supplemental 1C). T-DNA insertions were verified by PCR amplification using primers flanking genomic sequence of AtWDR26 and located in the T-DNA left boarder (LB). As shown in supplemental 1D, PCR amplification using the AtWDR26 flanking primer obtained approximate 3 kb genomic DNA fragment in the wild-type and using primers specific to AtWDR26 and LB gained truncated genomic DNA fragments in both KO lines (supplemental 1D). Furthermore, RT-PCR was conducted to verify gene knockout of AtWDR26 in both KO lines (supplemental 1E). Seed germination of OE transgenic plants was examined under dark- and light grown condition. Our results showed that seed germination rates of the OE transgenic seeds were reduced under light grown condition (Fig. 3A). However, the dark grown seed germination exhibited no significant difference between the OE and wild-type plants (data no shown). Moreover, seedlings of KO lines exhibited greener and larger cotyledons under light grown condition, but without change in hypocotyl growth (Fig. 3B). Phenotypes of seedling growth in the KO lines resembled the typical physiological responses regulated by photoreceptors such as chloroplast accumulation in cotyledons [41,42]. However, seed germination rate of the KO lines was similar to that of wild-type under light grown condition (data not shown). 3.3. Role of AtWDR26 in abiotic stress response The AtWDR26 expression was induced by osmotic stress and salt stress (Fig. 2B). To verify the role of AtWDR26 in abiotic stress response, two transgenic lines, OE-1 and OE-5, were used to test for salt stress response. Two different salt stresses, 300 ␮M ZnSO4 and 150 mM NaCl, were tested in this study. Our results showed that the root growth of both OE seedlings exhibited an increased tolerance to 300 ␮M ZnSO4 (Fig. 4A). However, the root growth of OE-1 was significantly increased, but that of OE-5 was slightly increased under 150 mM NaCl salt stress (Fig. 4B). For osmotic stress, root growth of both OE lines was hypersensitive to the addition of 300 mM mannitol (Fig. 4C). However, root growth of the KO lines exhibited no significant alteration in response to the addition of ZnSO4 , NaCl, or mannitol (data not shown). However, seed

Table 1 Genes activated more than 10 folds by AtWDR26 overexpression. Acc. no.

Description

Folds*

AT4G11393 AT2G36255 AT3G59930 AT5G33355 AT1G57850 AT3G15450 AT2G33830 AT1G28330 AT4G25490 AT3G61940 AT5G56080 AT2G32270 AT1G10970 AT4G33020 AT2G32290 AT5G49360 AT2G19800 AT5G44440 AT3G47340 AT4G26530 AT1G15380 AT1G63030

Defensin-like protein (DEFL) Defensin-like protein (DEFL) Defensin-like protein (DEFL) Defensin-like protein (DEFL) Toll-interleukin-resistance (TIR) domain-protein Aluminium induced protein with YGL and LRDR motif Dormancy-associated family protein, DYL1 Dormancy-associated protein like, DYL1 C-repeat/DRE-binding factor (CBF1) MTPA1; zinc ion transporter Nicotianamine synthase (NAS2) Zinc ion transporter (ZIP3) Zinc transporter 4 (ZIP4) ZIP9; cation transporter Beta-amylase 6 (BAM6) BXL1 (Beta-xylosidase 1) MIOX2 (Myo-inositol oxygenase 2) FAD-binding domain-containing protein Asparagine synthase (ASN1) Fructose-bisphosphate aldolase Lactoylglutathione lyase family protein Dwarf- and delayed flowering (ddf2)

76 68 15 13 11 18 24 13 14 69 32 11 10 39 11 10 10 26 17 15 14 10

*

Induction folds.

germination of the KO lines exhibited hypersensitive response to 150 mM NaCl and 300 mM mannitol (Fig. 4D and E). These results suggest that increased AtWDR26 expression exerted positive and negative effects on seedling growth under ionic and osmotic stress, respectively. Furthermore, AtWDR26 is required for seed germination under ionic and osmotic stress conditions. 3.4. Role of AtWDR26 in hormone signaling pathways The AtWDR26 transcript was induced by multiple hormones (Fig. 2B). A root growth assay was used to validate the role of AtWDR26 in hormone signal transduction. Our results showed that increased the AtWDR26 expression resulted in increased sensitivity to various concentration of 2, 4-D (Fig. 5A). Moreover, root growth of OE transgenic lines also increased sensitivity to exogenous JA and ABA (Fig. 5B and C). Moreover, the hypocotyl growth of OE seedlings was inhibited to a greater extent in medium containing 5 ␮M ACC (Fig. 5D). However, root growth of KO lines exhibited no significant alteration in response to different hormones (data not shown). These results suggest that AtWDR26 may play a direct or indirect role in multiple hormone signal transduction pathways including auxin, JA, ET and ABA. 3.5. Transcriptome alterations by AtWDR26 overexpressing To further analyze the AtWDR26 function, transcriptome analysis was conducted in transgenic plants overexpressing AtWDR26. For this purpose, transgenic plants OE-1 exhibiting altered phenotypes in the light grown seed germination, sensitivity to salt and osmotic stress, and perception to hormones was selected for microarray analysis. A total of more than 348 Arabidopsis genes were up-regulated more than 3-fold in transgenic seedlings (see supplemental 2). Among these, genes involved in various metabolic pathways constituted the largest group of AtWDR26 activated genes (Fig. 6A). Genes involved in the chloroplast-related metabolism and cell wall biogenesis constituted the largest and second gene group in this up-regulated gene category (Fig. 6B). Gene group of the chloroplast-related metabolism included genes involved in the photosystem electron transfer reaction, CO2 assimilation, sugar metabolism and peroxisome-related cellular events (supplemental 1). Moreover, significant numbers of genes involved in lipid, secondary metabolite synthesis, and nitrogen

H.-w. Chuang et al. / Plant Science 241 (2015) 23–31 Table 2 Up-regulated genes involved in hormone signals in transgenic plant overexpressing AtWDR26. Acc. no.

Description

Folds

Auxin NM 119380 NM 114298 AT5G51470 AT4G22620 AT5G18010 AT1G29460 AT4G34790 AT4G13790 AT5G18030 AT5G18060 AT5G18080 AT4G12410 NM 126068

IAA29 NIT2 (nitrilase 2) Auxin-responsive GH3 family protein Auxin-responsive family protein Auxin-responsive protein Auxin-responsive protein Auxin-responsive family protein Auxin-responsive protein Auxin-responsive protein Auxin-responsive protein Auxin-responsive protein Auxin-responsive protein HB53 transcription factor

8.7 7.0 6.2 4.2 3.8 3.6 3.4 3.4 3.3 3.2 3.1 3.0 3.3

JA AT5G62350 AT5G62360 OPR3 JAZ7 DGL

Invertase/pectin methylesterase inhibitorn (FL5-2I22) Invertase/pectin methylesterase inhibitor family protein 12-Oxophytodienoate reductase (OPR3) JAZ7 (JASMONATE-ZIM-DOMAIN PROTEIN 7) DGL (DONGLE); triacylglycerol lipase (DGL)

3.2 7.8 3.9 7.1 8.0

GA AT1G74670 ATGA2OX4 ATGA2OX2

Gibberellin-responsive protein Gibberellin 2-beta-dioxygenase (ATGA2OX4) Gibberellin 2-beta-dioxygenase (ATGA2OX2)

4.2 3.2 4.7

29

binding factor 1 (CBF1), a subfamily of AP2 family protein [52], was strongly up-regulated in the transgenic plant overexpressing AtWDR26 (Table 1). Transcriptome analysis revealed that AtWDR26 strongly activated 3 zinc transporters (ZIPs) (Table 2). Members of ZIP family function in zinc ion uptake [53]. Furthermore, MTPA1, a member of the zinc transporter (ZAT) family, was induced to high levels in the transgenic plants (Table 1). MTPA1 is responsible for zinc ion tolerance in Arabidopsis halleri [54]. Nicotianamine synthase 2 (NAS2) participates in the synthesis of nicotianamine (NA), an important regulator for zinc and iron homeostasis [55]. The transcript of NAS2 was also strongly activated in the OE seedlings (Table 1). These results suggested that AtWDR26 overexpression strongly up-regulated genes involved in ion homeostasis. Approximate 6% of up-regulated gene belonged to genes related to hormone signaling pathway or hormone synthesis (Fig. 6A and supplemental 2). Among them, genes related to auxin signaling and synthesis consisted of a large proportion in this gene category, which included nitrilase 2 (NIT2) involved in auxin biosynthesis [56], auxin responsive gene IAA29 [57] (Table 2). Moreover, the second significant gene group in this category was related to JA signaling such as jasmonate-ZIM-domain protein 7 (JAZ7) involved in JA signal transduction [58] (Table 2). Transcriptome evidence indicated the involvement of AtWDR26 in multiple hormonal signaling pathways. These results showed that overexpression of AtWDR26 altered gene expression implicated in hormone signaling.

Cytokinin (CK) AT3G48100 ARR5 (Arabdopsis response regulator 5) AT1G19050 ARR7 (Response regulator 7)

3.0 3.4

Ethylene (ET) AT2G22810 ACS4 (1-aminocyclopropane-1-carboxylate synthase 4)

4.9

4. Discussion

ABA AT4G32810

3.5

AtWDR26 was previously isolated as an interacting protein of the AtRanBPM complex [28]. However, the biological role of this complex remains uncharacterized. The chloroplast signaling plays an important role in plant acclimation to high light condition [59]. This chloroplast retrograde signal is implicated in the crosstalk interaction between high light acclimation and immunity [23,60]. Moreover, the response to high light is accompanied with rapid up-regulation of expression of the AP2/ERF transcription factor family [61], and simultaneously increased tolerance to other abiotic stress factors including high temperature and water deficit [62], and perception to hormone signalings [63]. Physiological analysis of the AtWDR26 OE and KO lines confirmed the role of AtWDR26 in light, abiotic stress and light signaling. Transcriptome analysis provided additional evidence for the involvement of AtWDR26 in transcription regulation of biotic and abiotic stress, and chloroplast-related reactions. For example, AtWDR26 overexpression strongly up-regulated several members of DEFL genes. Moreover, up-regulated gene included transcription factor CBF1 functioning in cold and high light acclimation by regulating the chloroplastic H2 O2 -triggered retrograde signaling [60]. Additionally, genes involved in phytochrome-mediated signaling pathway such as PAR1 and FHL were up-regulated in transgenic overexpressing AtWDR26 (supplemental 2). Furthermore, FHL is a far-red-elongated hypocotyl (FHY)—like protein. Study has demonstrated involvement of FHY3 in the regulation of plant immunity by modulating chlorophyll biosynthesis [64]. Several members of ERF/AP2 family were up-regulated by AtWDR26 overexpression which included ERF6, ERF8 and RRTF, whose expression is an indicator of high light response [61]. Overexpression of AtWDR26 up-regulated several members of Plant U-box (PUB) ubiquitin ligases (supplemental 2). Among these, PUB12 is involved in the flagellin induced immunity response [65] and PUB22 targets proteins participating in pathogen-associated molecular patterns (PAMPs)-induced immunity [66]. Physiological and transcriptional evidence suggests that AtWDR26 might be a regulatory

CCD8 (carotenoid cleavage dioxygenase 8); oxidoreductase

assimilation were up-regulated by AtWDR26 overexpression (Fig. 6B). The AtWDR26 up-regulated metabolism-related genes resembled metabolism acclimation to high light that photosynthetic photon flux, metabolisms of sugar, lipid, nitrogen and secondary metabolites, and cell wall lignification was altered [43–45]. Photoreceptor-mediated physiological response crosstalks with regulatory components implicated in response to high light acclimation [46]. Our results showed that overexpression of AtWDR26 resulted in the up-regulation of genes related to developmental process related to photoreceptors such as FARRED-elongated hypocotyl-like (FHL), Phy rapidly regulated 1 (PAR1), CONSTANS-like protein-related, and phototropic responsive NPH3 family gene (supplemental 1). A large number of up-regulated genes were implicated in various cellular events related to stress response which included genes involved in redox reactions and response to biotic and abiotic stress (Fig. 6A and C). Among the biotic stress-related genes, 4 members of the defensin-like (DEFL) gene family were strongly up-regulated (Table 1). Among them, the DEFL gene (AT4G11393) was induced to the highest levels among all AtWDR26-up-regulated genes. The defensin gene family plays a particular role in plant disease resistance [47,48]. Antimicrobial activity of the DEFL family has been demonstrated [49]. AtWDR26 overexpression also up-regulated a large number of genes exhibiting function or predicted function in plant response to various abiotic stress including senescence, cold, water stress, and heat stress (supplemental 2). A large number of transcription factors were up-regulated in the transgenic plant overexpressing AtWDR26 (Fig. 6A). Among them, AP2/ERF transcription factor family constituted the largest proportion of the up-regulated transcription factors (Fig. 6D and supplemental 2). This gene family plays a significant role in plant response to both biotic and abiotic stress [50,51]. Among them, C-REPEAT/DRE

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component involved in the crosstalk between high light acclimation and immunity response. ROS-induced signaling crosstalks with multiple hormone responses including ET, JA, and ABA [67]. Moreover, defects in antioxidative capacity like thioredoxin resulted in increased oxidative stress and lead to altered auxin homeostasis [68]. Seedling growth of OE and KO lines of AtWDR26 exhibited altered sensitivity to several hormone signals including auxin, JA, ET, and ABA. Human WDR26 plays a role in regulating the death of neural cells by controlling the H2 O2 level [27]. Moreover, transcriptome evidence indicated that the AtWDR26-up-regulated genes included the thioredoxin family genes, redox responsive transcription factor (RRTF) involved in the regulatory network of photosynthetic stress [69] and UPBEAT1 (UPB1) controlling the ROS homeostasis in the root elongation zone [70]. Hence, it is plausible that the altered response to multiple hormone signals in the OE and KO lines of AtWDR26 occurred due to changes in the ROS regulatory network which leads to alterations in multiple hormone signaling pathways in Arabidoposis. 5. Conclusions Transcriptional and phenotypic evidence supports the role of AtWDR26 in controlling cellular signaling implicated in the regulation of high light acclimation and defense response to biotic and abiotic stresses. AtWDR26 may modulate the ROS homeostasis to play an indirect role in regulating the signal transduction of multiple hormones, including auxin, ET, JA and ABA.

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Acknowledgments We would like to thank the ABRC for providing us with seeds of T-DNA insertion lines of AtWDR26. 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.2015.09. 024.

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