Plant Physiology and Biochemistry 77 (2014) 7e14

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Research article

Heterologous expression of the gourd E3 ubiquitin ligase gene LsRZF1 compromises the drought stress tolerance in Arabidopsis thaliana Ji-Hee Min a, Hyun-Woo Ju a, Kwang-Yeol Yang a, Jung-Sung Chung b, Baik-Ho Cho a, Cheol Soo Kim a, * a b

Department of Plant Biotechnology, Chonnam National University, Gwangju 500-757, Republic of Korea Department of Agronomy, Gyeongsang National University, Jinju 660-701, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 December 2013 Accepted 18 January 2014 Available online 25 January 2014

Protein ubiquitination is one of the major regulatory processes used by eukaryotic cells. The ubiquitin E3 ligase acts as a main determinant of substrate specificity. However, the precise roles of E3 ligase in plants to drought stress are poorly understood. In this study, a gourd family (Lagenaria siceraria) ortholog of Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1) gene, designated LsRZF1, was identified and characterized. LsRZF1 was reduced by abscisic acid (ABA), osmotic stress, and drought conditions. Compared to wild type, transgenic Arabidopsis plants ectopic expressing LsRZF1 were hypersensitive to ABA and osmotic stress during early seedling development, indicating that LsRZF1 negatively regulates droughtmediated control of early seedling development. Moreover, the ectopic expression of the LsRZF1 gene was very influential in drought sensitive parameters including proline content, water loss, and the expression of dehydration stress-related genes. Furthermore, ubiquitin E3 ligase activity and genetic data indicate that AtRZF1 and LsRZF1 function in similar pathway to control proline metabolism in Arabidopsis under drought condition. Together, these results suggest that the E3 ligase LsRZF1 is an important regulator of water deficit stress during early seedling development. Crown Copyright Ó 2014 Published by Elsevier Masson SAS. All rights reserved.

Keywords: Abscisic acid Dehydration E3 ubiquitin ligase Gourd family LsRZF1 RING-H2 zinc finger protein

1. Introduction Gourd family (Lagenaria siceraria) is one of the most important agricultural crops in the world. Water deficit is the major limiting factor for crop productivity. To improve gourd tolerance to drought stress, identification of abiotic stress tolerance-related genes is important. Proline accumulation is a common physiological response in higher plants exposed to drought and salt stress (Delauney and Verma, 1993). Proline is considered to be an important osmolyte that acts as a molecular chaperone to stabilize the structure of proteins, as well as a regulator of cellular redox potential and an antioxidant controlling free radical levels (Hare et al., 1999). Plants that are challenged by drought and salt stress recruit ABA as an endogenous signal that initiates adaptive responses (Zhu, 2002). During late embryogenesis, ABA promotes the acquisition of desiccation tolerance and seed dormancy, and inhibits seed

Abbreviations: MS, Murashige and Skoog; qPCR, quantitative real-time polymerase chain reaction; WT, wild type; ZF, zinc finger. * Corresponding author. Tel.: þ82 62 530 2182; fax: þ82 62 530 2079. E-mail address: [email protected] (C.S. Kim).

germination (Koornneef et al., 2002; Song et al., 2005). The molecular mechanisms underlying ABA-mediated plant tolerance to drought stress are still not fully understood because of the complex nature of the plant response to ABA signaling and drought stress. However, the study of stress-responsive transcription factors has been one of the foci in studies on drought stress tolerance (Fujita et al., 2011). Protein ubiquitination is an important post-translational modification process that is employed by eukaryotes to regulate diverse cellular and developmental processes (Dye and Schulman, 2007). In higher plants, ubiquitinated proteins are involved in abiotic or biotic stress response, hormone response, cell cycle progression and cell differentiation (Craig et al., 2009; Santner and Estelle, 2009; Marrocco et al., 2010; Ryu et al., 2010). RING (for Really Interesting New Gene) motif-containing E3 ubiquitin ligase comprises one of the largest gene families in the plant genome. The Cys-rich RING zinc finger was first described in the early 1990s (Freemont et al., 1991). It was defined as a linear series of conserved Cys and His residues (C3HC/HC3) that bind two zinc atoms in a cross brace arrangement. RING zinc fingers can be divided into two types, C3HC4 (RING-HC) and C3H2C3 (RING-H2), depending on presence of either a Cys or a His residue in the fifth position of the motif (Freemont, 2000). Recently, a number of Arabidopsis RING E3

0981-9428/$ e see front matter Crown Copyright Ó 2014 Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2014.01.010

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ubiquitin ligases were shown to be involved in various cellular processes, such as auxin signaling, ABA signaling, brassinosteroid response, seed germination, seedling development, adaptive pathway to nitrogen limitation, and sugar responses (Santner and Estelle, 2009; Stone et al., 2006; Peng et al., 2007; Bu et al., 2009; Huang et al., 2010). In particular, RING proteins play a key role in the response to environmental stimuli. For example, they participate in photomorphogenesis, defense signaling, senescence, and tolerance mechanisms against cold, drought, salt and osmotic stress (Fujita et al., 2011; Craig et al., 2009; Yan et al., 2003; Smirnova et al., 2011). Recently, we described the functional characterization of a C3H2C3-type RING-H2 zinc finger protein, designated Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1) (Ju et al., 2013). Functional studies demonstrated that AtRZF1 participates negatively in proline production under drought condition. Water deficit response assays indicated that, while the atrzf1 mutant was less sensitive to drought, AtRZF1-overexpressing plants were more sensitive, suggesting that AtRZF1 negatively regulates the drought response during early seedling development. Furthermore, our previous data reported that AtRZF1 is a functional ubiquitin E3 ligase (Ju et al., 2013). In this research, an ortholog of the AtRZF1 gene in the gourd species L. siceraria, LsRZF1, was cloned to examine the role of LsRZF1 by expression in Arabidopsis. The gene was reduced in response to drought stress and ABA phytohormone. The response of constitutive expression, leading to both complementation and ectopic expression of LsRZF1 in the atrzf1 mutant and transgenic Arabidopsis plants, respectively, against different abiotic stresses was studied. LsRZF1-ectopic expressing plants were hypersensitive to osmotic stress in terms of cotyledon development due to the reduction in the contents of proline and regulated the expression of stress-related genes. The physiological processes of the drought induced phenotype began faster in the LsRZF1-ectopic expressing lines than in the wild type (WT) and atrzf1 complementation plants. Additionally, ubiquitin E3 ligase activity and genetic data indicated that AtRZF1 and LsRZF1 function in a similar pathway to control proline metabolism in Arabidopsis under drought condition. 2. Materials and methods 2.1. Plant materials, growth conditions and stress induction Arabidopsis (Col-0) or gourd (L. siceraria) 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 osmotic stress by submersion of 14-day-old gourd seedlings in a solution containing 400 mM mannitol. Samples were obtained at 0 and 24 h of osmotic stress. For ABA challenge, 1-week-old gourd seedlings were submerged in a solution containing 150 mM ABA and sampled at 0 and 6 h. For drought stress, seedling plants were grown in pots with normal watering every 3 days. After 1 week, the plants were divided into two groups for stress treatments. One group was subjected to drought stress by withholding water for 7 days, and a control group was watered normally. In each case, the retrieved seedlings were promptly frozen in liquid nitrogen and stored at 80  C. 2.2. Quantitative real-time (RT)-PCR Quantitative RT-PCR (qPCR) 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 ABA- or dehydration stress-treated gourd and Arabidopsis seedlings using a RNeasy Plant Mini kit (Qiagen). qPCR was carried out using

the SensiMix One-Step kit (Quantance, London, UK). Arabidopsis Actin8 or Lagenaria siceraria Actin7 (LsACT7) was used as the internal control. Quantitative analysis was carried out using the Delta Delta CT method (Livak and Schmittgen, 2001). Each sample was run in three independent experiments. The reaction primers utilized are shown in Supplemental Table S1. 2.3. In vitro self-ubiquitination assay Full-length LsRZF1 cDNA was amplified using the following primers: 50 -GCGAATTCATGTCAGCTGGTCGGAACA-30 (EcoRI site underlined) and 50 -GCCTGCAGTGGTGAATTAGTGTATCGA-30 (PstI site underlined). PCR products were cleaved with EcoRI and PstI and inserted into a pMAL P2x vector (New England BioLabs, Beverly, MA, USA). This plasmid was expressed in Escherichia coli (E. coli) strain BL21 and purified by affinity chromatography using amylase resin (New England BioLabs). In vitro self-ubiquitination assay was carried out with an auto-ubiquitinylation kit (Enzo Life Sciences, Farmingdale, NY, USA). Reaction samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) using a semi-dry transfer cell (Bio-Rad, Hercules, CA, USA). Immunoblots were performed with anti-Ub antibody (Enzo Life Sciences) as described by the manufacturer’s product manual. 2.4. Ectopic expression construct of LsRZF1 Total RNA was isolated from gourd leaves using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription-PCR was employed to obtain full-length LsRZF1 cDNA. The generated product was then cloned into the pGEM T-easy vector for DNA sequence analysis. The reverse transcription-PCR primers were ForBI 50 GCGGATCCATGTCAGCTGGTCGGAACA-30 (BamHI site underlined) and RevSI 50 -GCGAGCTCTCATGGTGAATTAGTGTAT-30 (SacI site underlined) based on the sequence information contained in a cDNA database (http://www.icugi.org). Amplification proceeded for 35 cycles as follows: 94  C, 30 s; 55  C, 30 s; and 72  C, 1 min. The PCR-amplified products were double digested with BamHI and SacI, and directionally cloned into the plant expression vector pCAMBIA1301. The resultant construct was introduced into Arabidopsis tumefaciens strain GV3101 via in planta vacuum infiltration (Bechtold and Pelletier, 1998). Homozygous lines (T3 generation) from 16 independent transformants were obtained, and two lines (OX18-2 and OX32-5) with high transgene expression levels were selected for phenotypic characterization. In addition, ectopic expression of LsRZF1 in atrzf1 mutant plants was confirmed by RTPCR analysis. Kanamycin resistance of the T2 generation from these two LsRZF1-ectopic expressing and one atrzf1/LsRZF1 complementation selected lines was segregated as a single locus. 2.5. Extraction of RNA and RT-PCR Total RNA was extracted from the frozen samples using a Plant RNeasy extraction kit (Qiagen, Valencia, CA, USA). To remove any residual genomic DNA in the preparation, the RNA was treated with RNase-free DNase I in accordance with the manufacturer’s instructions (Qiagen). The concentration of RNA was quantified accurately via spectrophotometric measurements, and 5 mg of total RNA was separated on a 1.2% formaldehyde agarose gel to check their concentration and monitor their integrity. RT-PCR was employed to measure the levels of LsRZF1 expression in WT, atrzf1/ LsRZF1 complementation, and ectopic expression transgenic plants. Five hundred nanograms of total RNA was used in the RT-PCR reaction, together with the following primers: for LsRZF1, forward (50 -

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GCGGATCCATGTCAGCTGGTCGGAACA-30 ) and reverse (50 GCGAGCTCTCATGGTGAATTAGTGTAT-30 ); for Actin8, forward (50 TGCCTATCTACGAGGGTTTC-30 ) and reverse (50 GTCCGTCGGGTAATTCATAG-30 ). After 28 PCR cycles of amplification, 20 mL of each RT-PCR product were loaded onto a 1.2% (w/v) agarose gel to visualize the amplified DNAs. Each cDNA was synthesized using 2 mg of total RNA with a RevertAid first-strand cDNA synthesis kit (Fermentas, Burlington, Ontario, Canada). Amplification proceeded for 28 cycles as follows: 94  C, 30 s; 55  C, 30 s; and 72  C, 1 min. Primers used in RT-PCR analyses are listed in Supplemental Table S1. RT-PCR products were separated on 1% agarose gels by electrophoresis and imaged on a Gel-doc XR (Bio-Rad, Hercules, CA, USA). 2.6. Phenotype analysis and stress tests For the osmotic stress tests, seeds were sown on MS medium supplemented with 400 mM mannitol and permitted to grow in a growth chamber. Cotyledon greening of each seedling was measured at 10 days after germination. Experiments were conducted in triplicate for each line (50 seeds each). For the ABA cotyledon greening tests, seeds were sown on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 1 mM ABA and permitted to grow in a growth chamber. Cotyledon greening of each seedling was measured at 7 or 10 days. Experiments were conducted in triplicate for each line (50 seeds each). 2.7. Relative water content measurement For relative water content values, detached rosette leaves were placed on open lid-Petri dishes at room temperature with 60% humidity under dim light. The weights of rosette leaves were measured at various times. Leaf water content was expressed as the percent of initial fresh weight.

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Arabidopsis genome, and its biochemical functions have reported as one of the negative ubiquitin E3 ligase of stress responses (Ju et al., 2013). In an effort to gain an insight into the function of the RING-H2 zinc finger gene in plants, we attempted to isolate the gene in gourd species L. siceraria that encodes for sequences similar to the isolated AtRZF1. The isolated cDNA sequence comprised 918 bp and encoded 305 amino acids with a calculated molecular weight of 33.5 kDa. The protein harbored a predicted zinc finger domain as shown by software program (http://myhits.isb-sib.ch) (Fig. 1A). The deduced amino acid sequence displayed a considerable degree of homology with already identified AtRZF1 (Fig. 1B). The L. siceraria gene was therefore designated as LsRZF1. Overall homology values of 59% identity and 66% similarity were observed between LsRZF1 and AtRZF1 proteins. LsRZF1 contains a single RING domain in its central region that is 83% identical to the corresponding region of AtRZF1 proteins. Since the Cys-x2-Cys-x14-Cys-x1-His-x2-His-x2Cys-x10-Cys-x2-Cys sequence is well conserved in the 41 amino acid RING motif (Fig. 1C) (Jensen et al., 1998), LsRZF1 is a C3H2C3-type RING-H2 protein. A phylogenetic tree representing the distances between watermelon groups of sequences was built using a cluster algorithm (Supplementary Fig. S1). 3.2. LsRZF1 is down-regulated by abiotic stresses To obtain clues regarding the functions of LsRZF1, accumulation of LsRZF1 mRNA in 14-day-old gourd seedlings was assessed during drought and mannitol treatment using qPCR. As shown in Fig. 2, LsRZF1 transcripts were significantly reduced in response to drought and slightly declined during osmotic stress. LsRZF1 mRNA was reduced 5.1- and 1.5-fold by drought and mannitol treatments, respectively (Fig. 2). These results suggest that LsRZF1 is regulated by water deficit stresses. 3.3. LsRZF1 exhibits in vitro ubiquitin E3 ligase activity

2.8. Proline content determination Proline contents were measured as previously described (Bates et al., 1973). Briefly, proline was extracted from 100 mg of plant leaves by grinding in 1 ml of 3% sulphosalicylic acid. Two hundred microliters of extract was reacted with 100 ml of the ninhydrin reagent mixture (80% glacial acetic acid, 6.8% phosphoric acid, and 70.17 mM ninhydrin) for 60 min at 100  C. An ice bath was used to terminate the reaction. The reaction mixture was extracted with 200 ml of toluene and vortexed. Absorbance of the toluene layer was read at 520 nm in a UV/VIS spectrophotometer (JASCO, Tokyo, Japan). Proline concentration was determined from a standard curve, and calculated on a fresh weight (FW) basis as follows: [(ng proline/ml  ml extraction buffer)/115.5 ng nmol]/g sample ¼ nmol proline/g FW material. 2.9. Statistical analysis Statistical analyses were performed using the software in Excel and SPSS. Analysis of variance was used to compare the statistical difference based on Student’s t-test, at a significant level of 0.01 < P < 0.05 or P < 0.01. 3. Results 3.1. Identification and amino acid sequence analysis of the LsRZF1 gene Several lines of evidence show that AtRZF1 belongs to the C3H2C3-type RING-H2 zinc finger gene family in the complete

The AtRZF1 protein is a member of the C3H2C3-type RING-H2 protein (Fig. 1C) and has previously been tested for E3 ligase activity (Ju et al., 2013). Thus, it was of interest to test the ability of LsRZF1 to function as an E3 ligase in the ubiquitination process. Toward this end, LsRZF1 was tested for E3 ligase activity using in vitro assays (Fig. 3). Recombinant MBP-LsRZF1 protein was produced in E. coli and affinity purified using amylase resin. In the presence of E1 and E2, ubiquitinated MBP-LsRZF1 proteins were detected by immunoblot analysis using anti-Ub (Fig. 3) antibody. In the absence of either E1 or E2, the ubiquitination activity was not observed with MBP-LsRZF1. As shown in Fig. 3, high-molecularmass ubiquitinated bands were produced by LsRZF1, indicating that LsRZF1 had Ub E3 ligase activity in vitro. MBP-AtRZF1 protein was used as positive control for in vitro ubiquitin E3 ligase activity. 3.4. Ectopic expression of LsRZF1 confers high sensitivity to osmotic stress To assess its function in vivo, LsRZF1 ectopic expression was induced in Arabidopsis under the control of the 35S promoter. Sixteen homozygous lines (T3 generation) were obtained. Two lines (OX18-2 and OX32-5) with high levels of transgene expression (Fig. 4A) were selected for phenotypic characterization. In an effort to further evaluate the function of LsRZF1 in Arabidopsis, a complementation test was done. LsRZF1 gene was ectopically expressed in the atrzf1 mutant. An independent complementation line was selected and confirmed by RT-PCR. RT-PCR analysis revealed that LsRZF1 transgene was clearly expressed in atrzf1 complementation T3 transgenic plants (Fig. 4A). This T3

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Fig. 1. Structural features of the LsRZF1 protein. (A) The structure of the conserved regions of the LsRZF1 protein. The primary structure harbors RING-H2 zinc finger motif site (177e 217) is shown in the black box. (B) Predicted amino acid sequence of LsRZF1 and its comparison with related sequences. Shown are the sequences of LsRZF1 and AtRZF1 (At3g56580). Black and gray shading indicates identical and similar amino acids, respectively. Gaps were introduced to optimize the alignment. (C) Alignment of C3H2C3-type RING motif deduced amino acid sequences of LsRZF1 and AtRZF1 from different plant species. Shown are the sequences of LsRZF1 and AtRZF1 (At3g56580). Black and gray shading indicate identical and similar amino acids, respectively.

complementation line was used to analyze stress-related phenotypes. To assess the effects of LsRZF1 expression on dehydration stress, seeds of WT, atrzf1, atrzf1/LsRZF1 and LsRZF1-ectopic expressing plants were germinated on MS medium. Macroscopically, there was little difference in terms of plant growth among the WT, atrzf1, atrzf1/LsRZF1 and LsRZF1-ectopic expressing plants

Fig. 2. Expression of the LsRZF1 gene in gourd under water deficit stress. qPCR analyses of the expression of LsRZF1 involved in drought or mannitol response. Total RNA samples obtained from 14-day-old gourd seedlings treated with drought or 400 mM mannitol. The mean value of three technical replicates was normalized to the levels of LsACT7 mRNA, an internal control. Differences between the expression of LsRZF1 in gourd seedlings untreated () and treated (þ) with dehydration stress are significant at the P < 0.01 (**) levels.

(OX18-2, OX32-5) (Fig. S2A). The germination ratio among atrzf1, atrzf1/LsRZF1 and LsRZF1-ectopic expressing plants similar and not poor on MS medium (Fig. S2A). At 400 mannitol, approximately 30% of the WT leaves expanded

WT, was mM and

Fig. 3. E3 ubiquitin ligase activity of LsRZF1 in vitro. Purified MBP-LsRZF1 was incubated at 37  C for 1 h with E1, E2 (UbcH5a), ubiquitin (Ub), and ATP. Polyubiquitin chains were visualized with anti-ubiquitin antibody. Omission of E1 or E2 resulted in a loss of ubiquitination. MBP or MBP-AtRZF1 served as a negative or positive control, respectively. Numbers on the left indicate the molecular masses of marker proteins in kDa. (Ub)n, ubiquitinylated conjugates.

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Fig. 4. Osmotic stress sensitivity of LsRZF1-expressing transgenic plants. (A) Expression levels of LsRZF1 in WT, atrzf1/LsRZF1, and two independent transgenic lines ectopic expressing LsRZF1 (OX18-2 and OX32-5) were determined by RT-PCR using total RNA isolated from 2-week-old seedlings. Actin8 (ACT8) was used as an internal control in RT-PCR. (B) Osmotic stress effect on cotyledon greening. Seeds were sown on MS agar plates supplemented without () or with (þ) 400 mM mannitol and permitted to grow for 10 days, and seedlings with green cotyledons were counted (triplicates, n ¼ 50 each). Error bars represent standard deviations. Differences among WT, atrzf1 mutant, and transgenic plants grown in the same conditions are significant at the 0.05 > P > 0.01 (*) or the P < 0.01 (**) levels.

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(Fig. 5B). Under drought stress, a significant difference in proline content was observed among WT, atrzf1, atrzf1/LsRZF1 and LsRZF1-ectopic expressing plants. With regard to proline, the atrzf1 mutant exhibited higher levels than the WT, atrzf1/LsRZF1 and LsRZF1-ectopic expressing plants. The content of proline was slightly more induced by drought treatment in WT and atrzf1/ LsRZF1 plants than in the LsRZF1-ectopic expressing plants (Fig. 5B). These results suggested that LsRZF1 participates negatively in proline production under drought condition. Thus, LsRZF1 may be a similar function with AtRZF1 in the drought stress response. The expressions of the Delta1-Pyrroline-5-Carboxylate Synthase 1 (P5CS1), Alternative Oxidase 1a (AOX1a), and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) genes are induced by stress (Strizhov et al., 1997; Vanlerberghe et al., 2009; Tran et al., 2006). In plants, proline is synthesized from glutamate via D1pyrroline-5-carboxylate (P5C) by two successive reductions, which are catalyzed by P5CS and P5C reductase (P5CR). The P5CS1 gene is induced by ABA, salt, dehydration and cold stress conditions, while ERD15 is induced by salt and dehydration stresses. Additionally, the AOX1a gene is induced by drought, light, and oxidative stress. Fig. 5C shows that the transcript levels of stress-inducible genes including P5CS1, AOX1a, and ERD15 displayed slightly less induction in atrzf1/LsRZF1 and LsRZF1ectopic expressing OX18-2 and OX32-5 lines than in the WT and atrzf1 plants following drought treatment. However, expression of the three genes was more induced by drought treatment in the atrzf1 mutant lines than in the WT plants. Taken together, our expression data suggest that LsRZF1 acts negatively on drought stress-related genes. 3.6. ABA response of the LsRZF1-ectopic expressing lines

turned green 10 days after germination, as compared to less than 18% and 6e14% of the atrzf1/LsRZF1 and LsRZF1-ectopic expressing lines (OX18-2 and OX32-5), respectively (Fig. 4B). On the contrary, 80% of the atrzf1 mutant line remained alive at 10 days after germination (Figs. 4B and S2B). These results showed that the atrzf1/LsRZF1 and LsRZF1-ectopic expressing lines were more likely to be sensitive to osmotic stress than is the WT and the atrzf1 mutant. 3.5. Increased water loss and reduced proline content of LsRZF1ectopic expressing plants under drought stress To further evaluate the responses to drought stress, cut rosette water loss rates of the plants were estimated. To assess water loss from leaves, leaves of similar size, age, and positions on WT, atrzf1, atrzf1/LsRZF1 and LsRZF1-ectopic expressing plants were detached and measured for decreases in fresh weight, as described previously (Ju et al., 2013). After detachment, leaves from the atrzf1/ LsRZF1and LsRZF1-ectopic expressing plants exhibited higher loss of fresh weight than those from WT and atrzf1 plants under ambient conditions (Fig. 5A). The difference occurred within 20 min, and became more apparent following detachment. These results indicate that physiological processes of drought induced phenotype began faster in the LsRZF1-ectopic expressing line than in the WT and atrzf1 plants. Since AtRZF1 participates negatively in proline production under drought condition (Ju et al., 2013), we determined the proline content in rosette leaves of WT and transgenic plants. To assess whether there were differences in the accumulation of proline among WT, atrzf1, atrzf1/LsRZF1 and LsRZF1-ectopic expressing plants, the proline contents of leaves were determined at 10 days after drought treatment. Before stress, the contents of proline were at similarly low levels in all seedlings

There is strong evidence for cross-talk between drought stress and ABA signaling pathways (Savoure et al., 1997; Lim et al., 2010). As a signaling molecule, ABA is an important component of abiotic stress response pathways. We therefore decided to investigate the expression level of LsRZF1 in Arabidopsis after ABA treatment. To identify the association between LsRZF1 and ABA response, accumulation of LsRZF1 mRNA in 7-day-old gourd seedlings was assessed during ABA treatment using qPCR. As shown in Fig. 6A, qPCR results demonstrated that LsRZF1 mRNA showed 4.3-fold reduction by ABA treatment. To characterize the sensitivity of the LsRZF1-ectopic expressing lines to ABA stress, we evaluated the response to treatment with 1 mM ABA concentration. As the seeds of the WT, atrzf1, atrzf1/LsRZF1 and LsRZF1-ectopic expressing plants (OX18-2 and OX32-5) were germinated on MS medium containing 0 or 1 mM ABA, the relative reduction in the cotyledon greening of the atrzf1/LsRZF1 and LsRZF1-ectopic expressing lines in response to ABA treatment was more profound than was observed in the WT and atrzf1 plants at 10 days after germination (Figs. 6B and S3). In the WT plants, the cotyledon greening efficiency dropped to 26% relative to the untreated plants (100%), whereas the cotyledon greening efficiency of atrzf1/LsRZF1 and LsRZF1-ectopic expressing lines was 19% and 11e14%, respectively, of control levels with the experimental concentration of ABA (Fig. 6B). However, the cotyledon greening efficiency of atrzf1 mutant lines was reduced to 68% of that of untreated plants with the 1 mM ABA (Fig. 6B). These results showed that the LsRZF1-ectopic expressing lines were more likely to be sensitive to ABA response than is the WT and atrzf1 mutant plants. In addition, LsRZF1 can compensate the loss of AtRZF1 gene. This demonstrated that LsRZF1 is involved in the ABA-mediated drought stress response.

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Fig. 5. Measurement of water loss, leaf proline content, and expression levels of drought-responsive genes in WT, atrzf1, and LsRZF1-expressing transgenic plants. (A) Quantification of water loss in 14-day-old WT, atrzf1, atrzf1/LsRZF1, and two independent LsRZF1-ectopic expressing plants. Rosette leaves of the same developmental stages were excised and weighed at various time points after detachment. Water loss was calculated as the percentage of initial fresh weight. Data represent average values  SD of five leaves from each of seven replicates. The asterisk denotes a statistically significant difference compared with the WT [0.05 > P > 0.01 (*) or P < 0.01 (**)]. (B) Leaf proline content in WT, atrzf1, and LsRZF1-expressing transgenic plants. Light-grown 2-week-old plants were grown for 10 days with () or without (þ) watering. Leaf tissues were carefully excised after drought treatment, and used for measuring proline content. Error bars represent standard deviations. Differences among WT, mutant, and transgenic plants grown in the same conditions are significant at the 0.05 > P > 0.01 (*) or the P < 0.01 (**) levels. (C) Light-grown 2-week-old WT, atrzf1, and LsRZF1-expressing transgenic plants were further grown for 10 days with () or withholding (þ) water. Total RNA was obtained from treated plants and analyzed by qPCR using gene-specific primers in listed in Supplementary Table S1. Each bar indicates the induction fold of the P5CS1, AOX1a and ERD15 genes in response to drought stress as compared to the control treatment (normal condition). The mean value of three technical replicates was normalized to the levels of Actin8 mRNA, an internal control. Differences between the expression of P5CS1, AOX1a and ERD15 in Arabidopsis seedlings untreated and treated with drought stress are significant at the 0.05 > P > 0.01 (*) or the P < 0.01 (**) levels.

3.7. Effects of ABA and drought on stress-related genes The expressions of the Responsive to ABA 18 (RAB18), Responsive to Dessication 29A (RD29A) and RD29B genes are induced by stress (Savoure et al., 1997; Lim et al., 2010). In detail, RAB18, RD29A and RD29B are induced under drought, ABA and salt stress conditions. Fig. 7 shows that the transcript levels of stress-inducible genes including RAB18, RD29A and RD29B displayed less induction in atrzf1/LsRZF1 and LsRZF1-ectopic expressing OX18-2 and OX32-5 lines than in the WT and atrzf1 plants following ABA treatment. However, expression of the three genes was more induced by ABA treatment in the atrzf1 mutant lines than in the WT plants. Taken together, our expression data suggest that LsRZF1 regulates the expression of these stress marker genes under ABA stress condition. 4. Discussion In an effort to identify the novel components involved in water deficit response in gourd species L. siceraria, we have identified one

drought stress-reducible gene, LsRZF1, for functional analysis. The predicted LsRZF1 protein possessed C3H2C3-type RING-H2 zinc finger motif (Fig. 1), and phylogenetic analysis revealed indicated that LsRZF1 displays high homology with other RING-H2 zinc finger protein watermelon family genes (Fig. S1). RING motif-harboring proteins have been shown to work as ubiquitin E3 ligases (Stone et al., 2005). Our self-ubiquitination analyses demonstrate that the LsRZF1 protein is indeed an active E3 ligase based on the occurrence of autoubiquitination of the MBP-LsRZF1 fusion protein in the presence of the E1 and E2 enzymes (Fig. 3). Considering the great similarity between AtRZF1 and LsRZF1 (Fig. 1), it was appropriate to consider whether AtRZF1 and LsRZF1 perform a similar function in the drought stress response. As expected, atrzf1/LsRZF1 and LsRZF1-ectopic expressing lines were more sensitive to osmotic stress than the WT, whereas atrzf1 mutant plants displayed enhanced tolerance to osmotic stress (Fig. 4). These data suggest that the biological function of LsRZF1 is a similar with AtRZF1 in the drought stress response. Consequently, our study also demonstrates a distinct difference in water loss and proline contents among atrzf1/LsRZF1, LsRZF1-ectopic expressing

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Fig. 6. Expression of the LsRZF1 gene in gourd under ABA and ABA sensitivity of LsRZF1-expressing transgenic plants. (A) qPCR analyses of the expression of LsRZF1 involved in ABA response. Total RNA samples obtained from 7-day-old gourd seedlings treated without () or with (þ) 150 mM ABA. The mean value of three technical replicates was normalized to the levels of LsACT7 mRNA, an internal control. Differences between the expression of LsRZF1 in gourd seedlings untreated and treated with ABA stress are significant at the P < 0.01 (**) level. (B) Influence of LsRZF1-expressing transgenic lines on ABA sensitivity. Seeds were sown on MS agar plates supplemented without () or with (þ) 1 mM ABA and allowed to grow for 10 days, and seedlings with green cotyledons were counted (triplicates, n ¼ 50 each). Error bars represent standard deviations. Differences among WT, atrzf1, atrzf1/LsRZF1 and two LsRZF1-ectopic expressing plants grown in the same conditions are significant at the 0.05 > P > 0.01 (*) or the P < 0.01 (**) levels.

and atrzf1 mutant lines (Fig. 5A and B). The leaves of atrzf1/LsRZF1 and LsRZF1-ectopic expressing lines exhibited a significant increase in water loss under drought condition compared with WT and atrzf1 mutant leaves (Fig. 5A). In addition, the accumulation of proline in the atrzf1 mutant was greater than that in WT, atrzf1/ LsRZF1 and LsRZF1-ectopic expressing plants (Fig. 5B), which might suggest that LsRZF1 is a component responsible for induction of leaf drought sensitivity through the modulation of osmolytic components. In many plant species, proline accumulation under water deficit has been correlated with dehydration stress tolerance, and its concentration has been shown to be change of expression of genes that are involved in the abiotic stress response in plants (Hare et al., 1999; Ju et al., 2013; Liu and Zhu, 1997). Presently, the transcript levels of drought-inducible genes including P5CS1, AOX1a and ERD15 displayed stronger reduction in atrzf1/LsRZF1 and LsRZF1-

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ectopic expressing plants following drought treatment (Fig. 5C), suggesting that LsRZF1 can participate in the signaling required to regulate the expression of drought stress-related genes. Although plant RING motif-harboring ubiquitin E3 ligases have concerned much interest as they are involved in diverse cellular and developmental processes, there are only a few reports on the functional correlation between RING motif-harboring E3 ligases and ABA-mediated drought stress responses. Overexpression of AtAIRP1 (for ABA Insensitive RING Protein 1), encoding an Arabidopsis C3H2C3-type RING E3 ligase, resulted in drought tolerance through enhanced ABA sensitivity during germination (Ryu et al., 2010). Zhang et al. (2007) reported that increased expression of gene encoding the RING domain protein SDIR1 (for Salt- and Drought-Induced RING Finger 1) confer drought tolerance through ABA-dependent pathway. Overexpression of SDIR1 leads to ABA and salt hypersensitivity during seed germination and stomatal closure, resulting in tolerance to drought stress. Most recently, Cheng et al. (2012) reported that RGLG2 (for RING domain Ligase 2), a RING E3 ligase, negatively regulates the drought stress response by mediating AtERF53 (for Ethylene Response Factor 53) transcriptional activity in Arabidopsis. ABA accumulates in different plant tissues in response to water deficit and is believed to function as a signal for the initiation of acclimation to these stresses (Savoure et al., 1997; Lim et al., 2010). The LsRZF1 transcript level was clearly reduced in response to ABA (Fig. 6A). The response of constitutive expression leading to both complementation and overexpression of LsRZF1 in atrzf1 mutant and transgenic Arabidopsis plants, respectively, against ABA was studied. The atrzf1/LsRZF1 and LsRZF1-ectopic expressing plants were hypersensitive to ABA in terms of cotyledon greening (Figs. 6B and S3), supporting the notion that LsRZF1 is a component responsible for induction of drought sensitivity through the ABAdependent process. Furthermore, the transcript levels of ABAinducible genes including RAB18, RD29A, and RD29B displayed less induction in atrzf1/LsRZF1 and LsRZF1-ectopic expressing lines than in WT and atrzf1 plants following ABA treatment (Fig. 7). Several of the present results support the suggestion that LsRZF1 can regulate the drought stress response through an ABAdependent signal transduction pathway (Figs. 4e7). In conclusion, we present evidence that a functional ubiquitin E3 ligase, LsRZF1, is involved in drought-regulated seedling growth and act as a negative regulator of the water deficit stress response. In the future, transcriptional profiling analyses may provide additional insights into the mechanisms by which LsRZF1 exerts its function. Particularly interesting challenges are to identify LsRZF1interacting proteins by yeast two-hybrid screening approach. The results of this experiment will provide a better understanding of the cellular functions of LsRZF1 E3 ligase with regards to drought stress responses in plants. Author’s contributions The work presented here was carried out in collaboration between all authors. JH Min, HW Ju and CS Kim conceived the research theme. JH Min, HW Ju, KY Yang, JS Chung and BH Cho designed methods and experiments, performed the laboratory experiments and analyzed the data. JH Min and CS Kim wrote the manuscript.

Fig. 7. Expression levels of stress-regulated genes, RAB18, RD29A, and RD29B in Arabidopsis under ABA. mRNA levels were determined by qPCR using total RNA from 14day-old seedlings, which treated without () or with (þ) 100 mM ABA with gentle shaking for 4 h. Error bars indicate standard deviations of three independent biological samples. Arabidopsis Actin8 was used as the internal control. Differences among the expression of RAB18, RD29A and RD29B in 14-day-old Arabidopsis seedlings untreated and treated with ABA stress are significant at the 0.01 < P < 0.05 (*) or the P < 0.01 (**) levels.

Acknowledgments This work was supported in part by grants to C.S.K. from the Next-Generation BioGreen21 program (SSAC, PJ00949104) funded by the Rural Development Administration and a grant Korea Institute of Planning and Evaluation for Technology in Food,

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