JIPB

Journal of Integrative Plant Biology

The CONSTANS‐like 4 transcription factor, AtCOL4, positively regulates abiotic stress tolerance through an abscisic acid‐dependent manner in Arabidopsis 1

Department of Plant Biotechnology, Chonnam National University, Gwangju 500‐757, South Korea, 2Department of Agronomy, Gyeongsang National University, Jinju 660‐701, South Korea, 3Department of Rural and Biosystems Engineering, Chonnam National University, Gwangju 500‐757, South Korea. *Correspondence: [email protected]

Abstract The precise roles of the B‐box zinc finger family of transcription factors in plant stress are poorly understood. Functional analysis was performed on AtCOL4, an Arabidopsis thaliana L. CONSTANS‐like 4 protein that is a putative novel transcription factor, and which contains a predicted transcriptional activation domain. Analyses of an AtCOL4 promoter‐b‐ glucuronidase (GUS) construct revealed substantial GUS activity in whole seedlings. The expression of AtCOL4 was strongly induced by abscisic acid (ABA), salt, and osmotic stress. Mutation in atcol4 resulted in increased sensitivity to ABA and salt stress during seed germination and the cotyledon greening process. In contrast, AtCOL4‐overexpressing plants were less sensitive to ABA and salt stress compared to the wild type. Interestingly, in the presence of ABA or salt stress, the transcript levels of other ABA biosynthesis and stress‐related genes were enhanced induction in AtCOL4‐overexpressing and

INTRODUCTION A better understanding of the mechanisms underlying plant responses to abiotic stress, including salinity and dehydration, is essential to address current agronomic problems. The detrimental effects of salinity occur as the result of osmotic stress, interruption of metabolic activities by ionic excesses and imbalances, and interference of salt ions with the uptake of essential macro‐ and micronutrients (Tester and Davenport 2003). These adverse effects manifest in the inhibition of germination, reduction of growth, and developmental disturbances (Verslues et al. 2006). The phytohormone ABA is the central regulator of abiotic stress tolerance in plants, and coordinates a complex regulatory network enabling plants to cope with the stress (Cutler et al. 2010). During the late stages of embryogenesis, ABA promotes the acquisition of desiccation tolerance and seed dormancy, while inhibiting seed germination (Koornneef et al. 2002). A large body of evidence supports the notion that the transcriptional signaling cascade is comprised of a complex signaling network between abiotic stress and ABA signaling pathways in plants (Yamaguchi‐Shinozaki and Shinozaki 2006; Xu et al. 2011; Zhang et al. 2012). Evidently, transcription factors (TFs) perform crucial functions in these processes. The www.jipb.net

WT plants, rather than in the atcol4 mutant. Thus, AtCOL4 is involved in ABA and salt stress response through the ABA‐ dependent signaling pathway. Taken together, these findings provide compelling evidence that AtCOL4 is an important regulator for plant tolerance to abiotic stress. Keywords: Abscisic acid; B‐box zinc finger family; transcription factor; salt stress Citation: Min JH, Chung JS, Lee KH, Kim CS (2014) The CONSTANS‐like 4 transcription factor, AtCOL4, positively regulates abiotic stress tolerance through an abscisic acid‐dependent manner in Arabidopsis. J Integr Plant Biol XX: XXX–XXX. doi: 10.1111/jipb.12246 Edited by: Jianming Li, University of Michigan, USA Received Apr. 5, 2014; Accepted Jul. 28, 2014 Available online on Jul. 29, 2014 at www.wileyonlinelibrary.com/ journal/jipb © 2014 Institute of Botany, Chinese Academy of Sciences

Arabidopsis genome encodes more than 1,500 TFs, approximately 45% of which belong to families specific to plants (Riechmann et al. 2000). CONSTANS/CONSTANS‐like (CO/COL) belongs to a family of zinc finger TFs with 17 members in Arabidopsis, and contains one or two B‐box zinc finger (ZF) regions at the N‐terminus, and a CCT (CO, COL, TOC1) domain at the C‐terminus (Robson et al. 2001). CONSTANS was the first B‐box protein to be identified from the late flowering phenotype of a co mutant in Arabidopsis and CO homologs have successfully been identified in a range of plant species (Putterill et al. 1995; Drobyazina and Khavkin 2011). In the past decade, the CO genes have been extensively studied. Their reported involvement in many molecular and genetic processes includes control of the photoperiod response and flowering time (Zhang et al. 2011), regulation of circadian rhythms (Salome et al. 2006), and light signal transduction (Zobell et al. 2005). COL genes also have been reported to be involved in the other photoperiodically regulated developmental processes, apart from playing a key role in the photoperiodic response to flowering (Datta et al. 2006; Holefors et al. 2009). An inspection of the current Arabidopsis databases revealed that the expression profiles of many other COL genes are diurnally oscillated (Blasing et al. 2005). Some COL genes have been characterized with XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

Research Article

Ji‐Hee Min1, Jung‐Sung Chung2, Kyeong‐Hwan Lee3 and Cheol Soo Kim1*

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reference to functions associated with the regulation of lateral root formation (Datta et al. 2006), branching phenotype, and shade avoidance response (Wang et al. 2013), and the fruit ripening and stress responses in banana fruit (Chen et al. 2012). These results clearly indicate that the CO and COL genes have a wide‐ranging influence on plant development. However, whether COL genes are involved in abiotic stress responses is largely unknown. Herein, we identified the physiological role of the disruption and overproduction of a nuclear‐localized B‐box ZF type CO‐like protein, Arabidopsis thaliana CO‐like 4 protein (AtCOL4), in the plant response to ABA and salinity. The overexpression of AtCOL4 in transgenic Arabidopsis plants conferred increased insensitivity toward ABA and salt stress during seedling growth, while the AtCOL4 T‐DNA mutant (atcol4) plant gained sensitivity to ABA and conditions of high salinity. We further showed that AtCOL4 influences the expression of the ABA biosynthetic genes during ABA exposure.

RESULTS Identification and schematic structure of AtCOL4 (At5g24930) CONSTANS was the first B‐box protein to be identified in Arabidopsis (Putterill et al. 1995). Since its discovery, 16 other

COL proteins containing one or two B‐box domains at the N‐ terminus and a CCT domain at the C‐terminus have been identified (Robson et al. 2001). At5g24930 was identified as a member of the B‐box ZF CO‐like gene family from the Arabidopsis genome (Robson et al. 2001). The A. thaliana gene was therefore designated as AtCO‐like 4 (AtCOL4). The present analysis determined that the AtCOL4 gene possesses a translated region of 1218 bp that encodes a protein containing 406 amino acids, with a calculated molecular weight of 44.7 kDa. On the basis of the amino acid sequence, the predicted protein possesses a nuclear localization signal (NLS) beginning at the N‐terminus, B1‐ and B2‐box ZF regions, and a CCT domain at the C‐terminus (Figure 1A). AtCOL4 is a nuclear‐localized protein To determine the subcellular localization of AtCOL4, we generated transgenic Arabidopsis plants that produced a green fluorescent protein (GFP)‐AtCOL4 fusion product under the control of a cauliflower mosaic virus 35S gene promoter. The majority of the GFP protein was located in the cytoplasm of the root cells with a weak GFP fluorescence signal (Figure 1B), while the fluorescence of the GFP‐AtCOL4 construct was quite strong in the nuclei of the root cells of the resulting transgenic seedlings (Figure 1B). These results confirmed the identity of AtCOL4 as a nuclear‐localized protein, which is consistent with its predicted function as a putative transcription factor.

Figure 1. Nuclear localization of AtCOL4 (A) Schematic representation of AtCOL4. The primary structure contains the following motifs, in order from the N‐terminus: a nuclear localization signal (NLS) (23–40), zinc finger (ZF) B1 (45–93), a B2 domain (94–135), and a CCT domain (339–381). (B) Seven day old transgenic seedlings grown on sterile medium were analyzed for green fluorescent protein (GFP) expression by confocal microscopy. B/W, black and white; GFP–AtCOL4, green fluorescence in the nuclei of the root cells of transgenic Arabidopsis. XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

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AtCOL4 roles in abiotic stress response AtCOL4 has transcriptional activation activity in the central domain To determine whether AtCOL4 could display transcriptional activation activity, full‐length cDNA of AtCOL4 was fused to the GAL4 DNA binding domain. The resulting construct, AtCOL4‐ 406 (Figure 2A), was introduced into yeast strain Y190. The AtCOL4‐406 protein was found to be solely capable of inducing lacZ expression in the yeast cells (Figure 2A), indicating the putative transcriptional activator function of the full‐length AtCOL4 protein. To further define the regions corresponding to the transcriptional activation domain, mutants were constructed by deleting portions of the AtCOL4 protein (Figure 2A). When part of the N‐terminus was deleted (construct AtCOL4‐306), a positive color reaction developed within 3 h with the X‐Gal filter assay (Figure 2A). This was also

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the case with the constructs AtCOL4‐206, AtCOL4‐300, and AtCOL4‐200, as a high level of lacZ expression was evident within 3 h (Figure 2A). However, when the central peptide was deleted, leaving only the C‐terminal region (construct AtCOL4‐ 106) or the N‐terminal region (construct AtCOL4‐100), no lacZ expression was evident, even upon overnight incubation (Figure 2A). This implicated the central domain as the crucial area for transcriptional activation activity. When both the N‐ and C‐terminal peptides were removed from the construct, as for AtCOL4‐406 (Figure 2A, construct AtCOL4‐Center), the high level of lacZ expression within 3 h confirmed the importance of the central peptide sequences (Figure 2A). As the X‐Gal filter assay does not provide a quantitative measure of the lacZ expression level, a second set of experiments was conducted to assay b‐galactosidase activity in the lysates of yeast cells

Figure 2. Structure of AtCOL4 deletion mutants in pBD‐GAL4 used for transactivation activity (A) The NH2‐ and COOH‐terminal deletion mutants were constructed by polymerase chain reaction. Self‐transactivation, as illustrated by the color reaction in the X‐Gal assay (shown on right), occurred within 3 h for AtCOL4‐406 (1–406), AtCOL4‐306 (100– 406), AtCOL4‐206 (200–406), AtCOL4‐300 (1–300), AtCOL4‐200 (1–200), and AtCOL4‐Center (100–300) constructs. Overnight incubation failed to produce a self‐activation reaction with the AtCOL4‐106 (300–406) and AtCOL4‐100 (1–100) constructs. The (þ) symbol indicates that a positive reaction in the X‐Gal assay was detected, and the () symbol indicates that no X‐Gal reaction was detected. (B) Assay of b‐galactosidase activity. The values shown are averages of three independent experiments. Error bars indicate standard deviations. The asterisk denotes a statistically significant difference compared with the pBD‐GAL4 vector (P < 0.01). www.jipb.net

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expressing these deletion constructs. Figure 2B shows the b‐galactosidase activity recovered from yeast cells expressing the AtCOL4 deletion proteins. Fundamentally, the results of this analysis were consistent with those obtained from the filter assay. Analysis of b‐glucuronidase activity To gain insight into the function of AtCOL4, its expression pattern was initially assessed by histochemical b‐glucuronidase (GUS) staining of Arabidopsis transgenic plants harboring the AtCOL4 promoter‐GUS fusion construct. Analysis of the transgenic plants revealed strong GUS activity in the young leaves and relatively weak activity in the cotyledon (Figure 3A, B). In the leaves, GUS staining was exhibited in the vascular system (Figure 3A, B). In flowers, it was observed in the tip region of the style, in the sepals, and in the veins of the petals (Figure 3C). In the anther, GUS activity was detected in the filament, and strongly detected in the pollen (Figure 3D). To quantitatively analyze AtCOL4 expression level during leaf development, a quantitative real‐time polymerase chain reaction (qPCR) assay was performed. The expression pattern of AtCOL4 during leaf development from 1 week after germination (WAG) to 4 WAG is shown in Figure 3E. AtCOL4 transcripts showed relatively higher levels in the leaves at the later stages of development. AtCOL4 gene is regulated by ABA, mannitol, and salt stress AtCOL4 is induced by salt, dehydration, and osmotic stress (https://www.genevestigator.com/gv/), although the mechanism(s) of induction remain unknown. In an effort to determine the in vivo functions of AtCOL4, the accumulation of AtCOL4 mRNA was assessed in 14 d old Arabidopsis seedlings exposed to ABA, mannitol, and salt, using qPCR. As shown in Figure 4, AtCOL4 transcripts were significantly upregulated in response to ABA (3–6 h), osmotic stress (400 mM mannitol for 6–12 h), and high‐salt stress (150 mM NaCl for 3 h). The qPCR results revealed that AtCOL4 mRNA level was increased 2‐ to 3.8‐fold and 5.7‐ to 6‐fold by ABA and osmotic stress, respectively (Figure 4). The AtCOL4 transcript level was increased 2.8‐fold in Arabidopsis seedlings within 3 h of salt treatment, but declined thereafter (Figure 4). The Responsive to desiccation 29B (RD29B), Responsive to ABA 18 (RAB18), and RD29A genes were used as positive controls for ABA, mannitol, and salt stress, respectively. The magnitude of stress induction of RD29A and RD29B was greater than that of AtCOL4, while induction of AtCOL4 in response to mannitol treatment was higher than that of RAB18 (Figure 4). Collectively, the data in Figure 4 indicate that AtCOL4 is subject to control by water deficit stress and by the stress hormone ABA. ABA and salinity stress response of the AtCOL4‐ overexpressing lines To investigate the in vivo function of AtCOL4, Arabidopsis plants overexpressing AtCOL4, under the control of the 35S promoter, were generated. Twelve homozygous lines (T3 generation) were obtained, and two lines (OX3‐2 and OX9‐5) exhibiting high levels of transgene expression (Figure 5A) were selected for phenotypic characterization. In an effort to further evaluate the function of AtCOL4 in Arabidopsis, the At5g24930‐ tagged T‐DNA insertion mutant SALK_003389 (Salk Institute, San Diego, CA, USA) was obtained. The T‐DNA inserted in intron XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

1 of the At5g24930 gene was verified via PCR and the cloning of the left T‐DNA border (Figure 5B and see Materials and Methods section). Once homozygosity had been established, the absence of AtCOL4 was verified by qPCR analysis (Figure 5A). The respective mutant was designated as atcol4. To assess the responses of the plants with different AtCOL4 expressions to ABA treatment, the seeds of the wild‐type (WT), atcol4, and AtCOL4‐overexpressing plants were first germinated on Murashige‐Skoog (MS) medium as a control (Murashige and Skoog 1962). The germination rate among WT, atcol4, and AtCOL4‐overexpressing plants were similar on the medium (Figures 5C, 6A). Additionally, the developmental processes or flowering time under long day conditions were not affected in the transformants and the mutant (data not shown). In order to investigate whether AtCOL4 is involved in seed germination in response to abiotic stress, we examined the germination phenotypes of WT, atcol4 mutant, and two overexpression lines (OX3‐2 and 9‐5) in the presence of either 1 mM ABA or 150 mM NaCl. As shown in Figure 5C, the germination rate of atcol4 seeds was significantly lower than that of WT seeds on the second day after sown on plates when exogenous ABA was applied, while the OX3‐2 and OX9‐5 lines exhibited significantly higher germination rates than the WT. This indicated that AtCOL4 likely controls seed germination through ABA signaling. In the presence of 150 mM NaCl, the atcol4 seeds still showed a significantly lower germination rate than the WT, while the germination of the AtCOL4‐overexpressing lines was less inhibited by salt stress compared to the WT (Figure 5C), implying that AtCOL4 was involved in seed germination in response to salt stress. To further correlate AtCOL4 function with plant insensitivity to ABA during the post‐germination stage, the seeds of the WT, atcol4, and AtCOL4‐overexpressing plants were germinated on MS medium containing 1 mM ABA. The cotyledon greening efficiency of the WT was only slightly below 32% at 10 d after germination. Only 23.6% of the atcol4 cotyledons expanded and turned green, compared to the 50.5%–66.7% observed in OX3‐2 and OX9‐5 (Figure 6B, E). These results indicated that the atcol4 mutant was more likely to be sensitive to exogenous ABA than the WT, while the AtCOL4‐overexpressing plants were more insensitive. To evaluate the responses of plants with different AtCOL4 expression levels to elevated salinity, the seeds of the WT, atcol4, and AtCOL4‐overexpressing plants were germinated in MS medium supplemented with 150 mM NaCl, then permitted to grow for 7 d prior to assessment of the cotyledon greening efficiency. Less cotyledon expansion and delay in turning green at 7 d after germination was revealed in all of the plants, compared to the MS control. At 150 mM NaCl, approximately 26.6% of the WT leaves expanded and turned green, compared to the more than 58.1%–63.3% of the OX3‐2 and OX9‐5 lines (Figure 6C, E). In contrast, less than 14% of the atcol4 mutant line remained alive at 7 d after germination (Figure 6C, E). These results suggest that the different greening rates of these plants, shown in Figure 6, are indeed due to their differences in stress tolerance rather than due to differences in germination. The extent of the salt stress‐induced damage was also evaluated by measuring the survival rate. Most of the WT and atcol4 mutant plants died, showing a low survival rate (26.5% for WT, 15.9% for atcol4 mutant), but 40.3% of the OX3‐2 www.jipb.net

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Figure 3. Expression of the AtCOL4 gene in Arabidopsis (A–D) AtCOL4 promoter‐glucuronidase (GUS) expression pattern in transgenic Arabidopsis plants. Glucuronidase staining in a (A) 7 and (B) 14 d old seedling plant. (C) GUS staining in a flower of a 4 week old transgenic plant. (D) Glucuronidase expression in the anther, with strongly detected expression in the pollen (arrow). (E) Quantitative polymerase chain reaction (qPCR) analysis of the expression of AtCOL4. RNA levels were determined by qPCR using total RNA isolated at the indicated ages. Error bars indicate standard deviations. The asterisk denotes a statistically significant difference compared with the 1 week old Arabidopsis seedling (0.01 < P < 0.05). www.jipb.net

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Figure 4. Expression of the AtCOL4 gene in Arabidopsis under abscisic acid (ABA), mannitol, or salt stress Quantitative polymerase chain reaction (qPCR) analysis of the expression changes of AtCOL4 induced in response to ABA, mannitol, or NaCl. Total RNA samples were obtained from 2 week old plants treated with 100 mM ABA, 400 mM mannitol, or 150 mM NaCl at the indicated times. Error bars indicate standard deviations of three independent biological samples. Arabidopsis Actin8 was used as the internal control. Significant differences between the expression of AtCOL4 in untreated 14 d old Arabidopsis seedlings versus those treated with various abiotic stresses were indicated, at the  0.01 < P < 0.05 or the  P < 0.01 levels. The RD29B, RAB18, or RD29A gene was used as a control for the ABA, mannitol or salt stress treatments, respectively.

and 46.3% of the OX9‐5 lines continued to grow (Figure 6D, F). These results indicated that high levels of expression of AtCOL4 could enhance plant salt tolerance. Effects of ABA and salt on ABA synthetic or stress‐related genes It has been relatively well established that the expressions of the ABA‐deficient 1 (ABA1), Nine‐cis‐epoxycarotenoid dioxygenase 3 (NCED3), ABA2, ABA3, RD29A, RD29B, and RAB18 genes are induced by stress (Nambara and Marion‐Poll 2005; Huang et al. 2008). The transcription levels of ABA biosynthetic genes are differentially regulated by external and endogenous signals. In particular, the expression of ABA1, NCED3, and ABA3 genes are induced by dehydration in Arabidopsis, whereas the expression of ABA2 is induced by the application of glucose that induces ABA accumulation (Cheng et al. 2002). Biochemical studies indicated that the ABA2 transcription is not induced upon osmotic stress in Arabidopsis (Cheng et al. 2002). RD29A, RD29B, and RAB18 are also induced under drought, ABA, and salt stress conditions (Abe et al. 1997; Nakashima et al. 2006). Figure 7 revealed that the transcript levels of ABA‐ and other stress‐inducible genes including ABA1, NCED3, ABA3, RD29A, RD29B, and RAB18, but not ABA2, were enhanced following induction in AtCOL4‐overexpressing and WT plants following ABA or salt treatment, rather than in atcol4 mutant plants, The expressions of the RD29A, RD29B, and RAB18 were slightly less induced in the atcol4 mutant than in the WT and AtCOL4‐overexpressing plants. Furthermore, the transcript levels of these four ABA synthetic genes were not significantly different between the control (H2O) and ABA treatment in atcol4 mutant plants. These observations support the notion that AtCOL4 regulates the expression of these ABA synthetic or stress marker genes under ABA and salt stress conditions. However, the expression levels of ABA1, ABA3, and RD29B in XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

WT and AtCOL4‐overexpressing plants seems to be very similar under ABA or salt stress. This likely indicates that the overexpression of AtCOL4 by itself is not sufficient for the induction of ABA synthetic or stress‐related genes, and may need additional components.

DISCUSSION This work demonstrates that AtCOL4 can function as an abiotic stress‐responsive transcriptional factor in planta. The transgenic plants overexpressing AtCOL4 showed salt hyposensitivity, while the atcol4 knock‐out mutant showed salt hypersensitivity during the seed germination and seedling growth assays (Figures 5, 6), supporting the notion that AtCOL4 is a component of resistance to the salt stress‐ triggered defective early developmental process. Constitutively expressed GFP‐AtCOL4 fusion proteins localized to the nuclei of transgenic root cells (Figure 1). Amino acid sequence analysis revealed two conserved AtCOL4 domains: a double B‐box zinc finger type domain, and a CCT domain (Figure 1). The best characterized member of the B‐box ZF family is the CO protein. The characteristic nature of CO has been well documented concerning its role in the photoperiodic control of flowering time (Onouchi et al. 2000). CONSTANS protein has double B‐box ZF domains at the N‐terminal region, and also has a common C‐terminal CCT motif (Robson et al. 2001). In Arabidopsis, there are many proteins that have the same structural design as CO; these proteins are collectively designated COL (Robson et al. 2001). The presence of multiple COL genes in the genome of Arabidopsis suggests that they may share redundant functions in the regulation of photoperiod flowering. For example, whereas AtCOL3 acts as a flowering repressor during short day and long day conditions, www.jipb.net

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Figure 5. Germination phenotype of the wild type (WT), atcol4 and AtCOL4‐overexpressing lines in response to abscisic acid (ABA) and NaCl treatment (A) Expression levels of AtCOL4 in WT, atcol, and two independent transgenic lines overexpressing AtCOL4 (OX3‐2 and OX9‐5) were determined by quantitative polymerase chain reaction (qPCR) using the total RNA isolated from 2 week old seedlings. Actin8 was used as an internal control in qPCR. (B) Scheme of the T‐DNA insertion in the AtCOL4 gene. Exons are depicted as black boxes, introns as black lines and the 50 ‐ and 30 ‐untranslated regions as gray boxes. The location of the T‐DNA insertion in the atcol4 allele was shown as a triangle under the gene diagram. (C) Seeds of WT, atcol4, and AtCOL4‐overexpressing lines were treated with 1 mM ABA or 150 mM NaCl, or untreated. Error bars represent standard deviations. Asterisks indicate the significance of the difference from the corresponding WT values determined by Student’s t‐test ( 0.01 < P < 0.05 or  P < 0.01). Three independent experiments were conducted, and similar results were obtained. MS, Murashige‐Skoog medium. AtCOL9 delays flowering during long days, while AtCOL5 can induce flowering during short days (Cheng and Wang 2005; Datta et al. 2006; Hassidim et al. 2009). AtCOL7 promotes branching in conditions of a high red light to far‐red light ratio, but enhances shade avoidance syndrome in low red light to far‐ red light ratio conditions in Arabidopsis (Wang et al. 2013). However, the characteristics and physiological roles of the products of many of the cloned plant COL genes remain unclear. Herein, the Arabidopsis CO‐like 4 protein, AtCOL4, which functions as a putative stress‐inducible transcription factor in salt stress, was described. Furthermore, AtCOL4‐overwww.jipb.net

expressing plants showed insensitivity to ABA in comparison to the WT, whereas the atcol4 mutant lines evidenced increased sensitivity to ABA in cotyledon greening (Figure 6). This implies that AtCOL4 is a crucial component in the regulation of ABA or ABA‐mediated stress signaling pathways in Arabidopsis. It was previously reported that ABA accumulates in different plant tissues in response to water deficit and salinity stresses, and it is believed to function as a signal for the initiation of acclimation to these stresses (Pla et al. 1993). This study showed that AtCOL4 displays self‐transcriptional activation activity (Figure 2). According to the results of the XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

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Figure 6. Influence of AtCOL4 transgenic lines on abscisic acid (ABA) and salt stress tolerance (A–C) Seeds were sown on Murashige–Skoog (MS) agar (A) without and (B) with 1 mM ABA or (C) 150 mM NaCl, and allowed to grow for 10 and 7 d, respectively. The photograph shows that AtCOL4‐overexpressing (OX3‐2 and OX9‐5) transgenic lines were greener than the wild‐type (WT) and atcol4 mutant plants under both ABA and salt stress conditions. (D) Phenotypic comparison of plant growth. Seeds were germinated and grown on MS medium containing 150 mM NaCl for 18 d. (E) Effect of ABA and NaCl treatment on cotyledon greening. Seeds were sown on MS agar plates, or MS supplemented with 1 mM ABA or 150 mM NaCl, and permitted to grow for 10 and 7 d, respectively. Seedlings with green cotyledons were then counted (triplicate determinations, n ¼ 50 each). Error bars represent standard deviations. Significant differences between WT, mutant and transgenic plants grown in the same conditions were indicated at the  0.05 > P > 0.01 or the  P < 0.01 levels. (F) Percentage of surviving plants in the salt tolerance assays. Seeds were germinated for 18 d on MS medium containing 150 mM NaCl, and the surviving plants were scored (triplicate, n ¼ 30 each). Error bars represent standard deviations. Student’s t‐test was calculated at the probability of  P < 0.01. X‐Gal filter assay and the b‐galactosidase activity data (Figure 2), the central domain of the AtCOL4 protein was indicated to be involved in transcriptional activation activity. The data also demonstrated a distinct difference in the expressions of ABA1, NCED3, ABA2, ABA3, Rd29A, Rd29B, and RAB18A genes between the AtCOL4‐overexpressing transgenic line and the atcol4 mutant line upon exposure to exogenous ABA and salt stress. Figure 7 provides evidence that the expressions of these genes, with the exception of ABA2, are more strongly induced in AtCOL4‐overexpressing and WT plants than in the atcol4 mutant after salt or ABA treatment, possibly implying that AtCOL4 is a regulator protecting against the salt‐ or ABA‐triggered defective developmental growth process. In summary, this study identified and physiologically characterized the gene involved in the abiotic stress process in Arabidopsis. This gene regulates anti‐abiotic stress ability via XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

the regulation of ABA or salinity signal transduction, although its physiological mechanisms have yet to be characterized completely. Further analyses, including the detection of interaction partners, may provide novel insights into AtCOL4‐ mediated abiotic stress tolerance. Overall, the physiological functions of AtCOL4 appear to be worthy of further elucidation.

MATERIALS AND METHODS Plant materials, growth conditions, and stress induction Arabidopsis thaliana L. plants were grown in growth chambers under intense light at 22 °C, 60% relative humidity, and a 16 h day length. The plants were challenged with salt by submersion of the whole 14 d old Arabidopsis seedlings in a solution containing 150 mM NaCl. Samples were obtained after 0, 3, and 6 h of salt stress. For the ABA treatment of www.jipb.net

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Figure 7. Expression of abscisic acid (ABA) synthesis or stress‐regulated genes in Arabidopsis under ABA and salt stress mRNA levels were determined by quantitative polymerase chain reaction (qPCR) using the total RNA from 2 week old wild type (WT), atcol4, and two independent AtCOL4‐overexpressing (OX3‐2, OX9‐5) seedlings, which were treated in 100 mM ABA or 150 mM NaCl with gentle shaking for 3 or 6 h, respectively. Each bar indicates the fold of induction of the ABA1, NCED3, ABA2, ABA3, RD29A, RD29B, and RAB18 genes in response to ABA or salt stress, compared to the control (H2O) treatment. The mean value of three technical replicates was normalized to the levels of Actin8 mRNA, an internal control. Error bar represents the standard deviation. Asterisks indicate the significance of the difference from the corresponding WT values, determined by Student’s t‐test ( 0.01 < P < 0.05 or the  P < 0.01).

Arabidopsis, the whole 14 d old seedlings were submerged in a solution containing 100 mM ABA and sampled at 0, 3, and 6 h. For the mannitol treatment of Arabidopsis, the whole 14 d old seedlings were submerged in a solution containing 400 mM mannitol and sampled at 0, 6, and 12 h. For measurement of leaf development, samples were prepared from leaves of Arabidopsis plants grown in soil at 1 (two rosette leaves of >1 mm in length), 2 (six rosette leaves of >1 mm in length), 3 (10 rosette leaves of >1 mm in length), and 4 (13 rosette www.jipb.net

leaves of >1 mm in length) WAG. In each case, the retrieved seedlings were promptly frozen in liquid nitrogen and stored at 80 °C. Constructs and generation of transgenic plants To generate the AtCOL4 promoter‐driven GUS construct, the 1.9 kb upstream genomic fragment from the AtCOL4 translation start codon was amplified by PCR, and then digested by HindIII and BamHI. The fragment was cloned into the binary XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

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vector pCAMBIA1391, resulting in a transcriptional fusion of the AtCOL4 promoter with the GUS coding region. The following primers were utilized for amplification of the AtCOL4 promoter: 50 ‐CCCAAGCTTTCTATTTGCTGCAACTACCT‐30 and 50 ‐CGGGATCCGTATCCATCTAGATCGAATG‐30 . For the subcellular localization analysis of the AtCOL4 protein, the AtCOL4 cDNA fragment was amplified using primers (Table S1) on the basis of the sequence information in the National Center for Biotechnology Information cDNA database (www.ncbi.nlm.nih.gov). The PCR product was inserted into the pEGAD vector, under the control of the constitutive 35S promoter, at the EcoRI and HindIII cloning sites. The nucleotide sequence of the new construct was confirmed by DNA sequencing. The above constructs were then introduced into the Agrobacterium tumefaciens strain GV3101, which was utilized for the transformation of Arabidopsis plants by vacuum infiltration (Bechotold and Pelletier 1998). Localization of GFP‐AtCOL4 fusion proteins in transgenic plants To detect the intracellular localization of GFP‐AtCOL4 fusion proteins in transgenic plants, root samples were mounted on microscope slides and observed using a FluoView1000 confocal microscope (Olympus, Tokyo, Japan). Confocal images were obtained and processed using the FV10‐ASW 1.7A computer software (Olympus, Tokyo, Japan). Construction of AtCOL4 deletion mutants for the analysis of transactivation activity in yeast cells For the analysis of transactivation activity, a subclone of each type of the AtCOL4 deletion cDNA was constructed via PCR amplification using primers harboring the EcoRI and PstI restriction endonuclease sites (Table S2). The coding sequences were inserted into the pBD‐GAL4 vector, and the clones obtained were confirmed by DNA sequence analysis. Deletion constructs were transformed into the Y190 yeast strain (MATa gal4 gal80 his3 trp1‐901 ade2‐101 ura3‐52 leu2‐3 leu2‐112 þ URA3::GAL >>lacZ LYS2::GAL(UAS) >>HIS3 cycr) by the lithium method (Schiestl and Gietz 1989), and the cells were selected on –Trp medium. Colonies were replated on –Trp medium, and employed in X‐Gal filter assays as previously described (Bai and Elledge 1996). To measure the strength of the X‐Gal activity from the AtCOL4 deletion constructs, a liquid b‐galactosidase assay using o‐nitrophenyl b‐D‐galactopyranoside as a substrate was conducted, as described by the manufacturer (Clontech, Mountain View, CA, USA). Analysis of GUS activity Histochemical staining for GUS activity in transgenic plants was conducted as described previously (Jefferson et al. 1987). Whole seedlings or various tissues were immersed in 1 mM 5‐ bromo‐4‐chloro‐3‐indolyl‐b‐glucuronic acid solution in 100 mM sodium phosphate, pH 7.0, 10 mM ethylenediaminetetraacetic acid, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% Triton X‐100, and incubated overnight at 37 °C. Chlorophyll was cleared from the plant tissues via immersion in 70% ethanol. Overexpression construct of AtCOL4 Total RNA was isolated from Arabidopsis leaves using Trizol reagent (Invitrogen, Carlsbad, CA, USA). To remove any XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

residual genomic DNA from the preparation, the RNA was treated with RNase‐free DNase I, in accordance with the manufacturer’s instructions (Qiagen, Valencia, CA, USA). The concentration of RNA was quantified accurately via spectrophotometric measurements, and each cDNA synthesis was carried out using 4 mg of total RNA with a RevertAid first‐ strand cDNA synthesis kit (Fermentas, Burlington, ON, Canada). Reverse transcription (RT)‐PCR was utilized to obtain full‐length AtCOL4 cDNA. The RT–PCR primers were as follows: forward, 50 ‐GCTCTAGAATGGACCCCACATGGATAGA‐30 and reverse, 50 ‐CGAGCTCCTAAAATGTAGGTACAAGTCC‐30 . Amplification proceeded for 35 cycles at 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1.5 min. The generated product was then cloned into the pGEM T‐easy vector (Promega, Madison, WI, USA) for DNA sequence analysis. The resulting plasmid was double‐digested with XbaI and SacI, and was then directionally cloned into the plant expression vector, pBI121. The resulting construct was introduced into the A. tumefaciens strain GV3101 via in planta vacuum infiltration. Homozygous lines (T3 generation) from 12 independent transformants were obtained, and two lines (OX3‐2 and OX9‐5) evidencing high levels of transgene expression were selected for phenotypic characterization. Kanamycin resistance of the T2 generation from these two selected lines was segregated as a single locus. AtCOL4 T‐DNA insertion line The AtCOL4 T‐DNA insertion line SALK_003389 (atcol4) was acquired from the Arabidopsis T‐DNA insertion collection of the Salk Institute (Alonso and Stepanova 2003). To select plants homozygous for the T‐DNA insertion, the gene‐ specific primers 50 ‐GCGAATTCATGTCAGCTGAGGAAGTTCC‐30 and 50 ‐GCCTGCAGCTAAAATGTAGGTACAAGTC‐30 (upstream and downstream, respectively) were utilized for the atcol4 line. Plants yielding no PCR products with the gene‐specific primers were subsequently tested for the presence of the T‐DNA insertion using the gene‐specific forward primer in combination with the T‐DNA left border specific primer, 50 ‐GCGTGGACCGCTTGCTGCACCT‐30 . Phenotype analysis and stress tests For the ABA cotyledon greening tests, seeds were sown on MS medium supplemented with 1 mM ABA and permitted to grow in a growth chamber. Cotyledon greening of each seedling was measured at 10 d. Experiments were conducted in triplicate for each line (50 seeds each). For the salt stress tests, seeds were sown on MS medium supplemented with 150 mM NaCl, grown in a growth chamber, and assessed for the percentage of cotyledon greening after 7 d. Experiments were conducted in triplicate for each line (50 seeds each). Quantitative real‐time PCR Quantitative real‐time PCR was carried out with a Rotor‐Gene 6000 quantitative PCR apparatus (Corbett Research, Mortlake, NSW, Australia), and the results were analyzed using RG6000 1.7 software (Corbett Research). Total RNA was extracted from the 14 d old Arabidopsis seedlings subjected to the various treatments using an RNeasy Plant Mini kit (Qiagen). Quantitative real‐time PCR was then carried out using the SensiMix One‐ Step kit (Quantance, London, UK). Arabidopsis Actin8 (ACT8) was used as the internal control. Quantitative analysis was carried out using the Delta Delta CT method (Livak and www.jipb.net

AtCOL4 roles in abiotic stress response Schmittgen 2001). Each sample was run in three independent experiments. The reaction primers utilized are shown in Table S1. Statistical analysis Statistical analyses were performed using the software in Excel and SPSS. ANOVA was used to compare the statistical difference based on Student’s t‐test, at a significance level of 0.01 < P < 0.05, or P < 0.01.

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Holefors A, Opseth L, Ree Rosnes AK, Ripel L, Snipen L, Fossdal CG, Olsen JE (2009) Identification of PaCOL1 and PaCOL2, two CONSTANS‐like genes showing decreased transcription levels preceding short day induced growth cessation in Norway spruce. Plant Physiol Biochem 47: 105–115 Huang D, Wu W, Abrams SR, Cutler AJ (2008) The relationship of drought‐related gene expression in Arabidopsis thaliana to hormonal and environmental factors. J Exp Bot 59: 2991–3007 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: b‐ glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907

ACKNOWLEDGEMENTS

Koornneef M, Bentsink L, Hilhorst H (2002) Seed dormancy and germination. Curr Opin Plant Biol 5: 33–36

This work was supported in part by a grant to C.S.K. from the Next‐Generation BioGreen21 program (SSAC, PJ00949104), funded by the Rural Development Administration, and by the Basic Science Research Program, funded by the Ministry of Education, Science and Technology of Korea (NRF‐2010‐ 0022026).

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real‐time quantitative PCR and the 2DDCT method. Methods 25: 402–408

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SUPPORTING INFORMATION Additional supporting information can be found in the online version of this article: Table S1. Gene‐specific primers used for quantitative reverse transcription polymerase chain reaction (RT–PCR) assay Table S2. Clones and primers for AtCOL4 deletion mutants

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The CONSTANS-like 4 transcription factor, AtCOL4, positively regulates abiotic stress tolerance through an abscisic acid-dependent manner in Arabidopsis.

The precise roles of the B-box zinc finger family of transcription factors in plant stress are poorly understood. Functional analysis was performed on...
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