Mol Biol Rep (2014) 41:5931–5941 DOI 10.1007/s11033-014-3468-z

Role of wheat trHb in nitric oxide scavenging Dae Yeon Kim • Min Jeong Hong • Yong Weon Seo

Received: 28 September 2013 / Accepted: 14 June 2014 / Published online: 1 July 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract We examined the role of wheat truncated-hemoglobin (TatrHb) in nitric oxide (NO) scavenging in transgenic Arabidopsis plants by assessing the response to an NO donor/ scavenger and salt stress. The degree of increase in Na? and decrease in K? levels in the transgenic plants were more than those in the wild-type plants, and the ratio of Na? to K? increased in the transgenic plants under salt stress. Endogenous NO increased dramatically in the salt-treated wild-type plants but not in the transgenic plants. Additionally, the maximum photosystem II quantum ratio of variable to maximum fluorescence (Fv/Fm) in transgenic plants decreased more significantly than that in the wild-type plants, indicating that the transgenic plants suffered more severe photosynthetic damage because of salt stress than that by the wild type. Similar results were observed in germination experiments by using Murashige and Skoog media containing 100 mM sodium chloride. The Fv/Fm decreased in the leaves of salt-treated transgenic plants, indicating that transgenic seeds were more sensitive to salt stress than that by the wild-type seeds. In addition, the negative effect on seed germination was more severe in transgenic plants than in the wild types under NaCl treatment conditions. The results support the hypothesis that plant trHb shares NO scavenging functions and characteristics with bacterial trHb. Keywords 4,5-Diaminofluorescein diacetate  Nitric oxide  Salt stress  Triticum aestivum  Truncated hemoglobin D. Y. Kim  Y. W. Seo (&) College of Life Science and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701, Korea e-mail: [email protected] M. J. Hong Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Korea

Introduction Plant hemoglobins (Hbs) were first discovered in soybean (Glycine max) root nodules [1] and are believed to exist in all plant species [2]. On the basis of phylogenetic and physiologic studies, plant Hbs are divided into class 1, class 2, and truncated (tr) Hbs [2–4]. The trHbs typically have 20–40 fewer amino acids than traditional Hbs, which contain the classically conserved globin fold [5, 6]. trHbs are present in a large number of microbial species and are characterized by a two-over-two a-helical packing instead of the classical three-over-three helical arrangement [7]. In contrast to other Hbs, trHbs are characterized by remarkable variability at the active site, particularly on the distal side of the heme pocket [6]. This feature may be related to their diverse physiological roles, including terminal oxidation [6], oxygen sensing, and scavenging for oxygen and nitric oxide (NO) [8]. On the basis of numerous in vivo and in vitro experimental observations, Bacillus subtilis trHbs have been shown to have extremely high oxygen affinity and a very slow rate of ligand release, which indicates that trHbs may act as oxygen transporters [9]. NO serves as a signal in plants and animals. In plants, it is involved in regulating growth [10], the signaling of abscisic acid-induced stomatal closure [11], and the expression of defense-related genes against pathogens and apoptosis/programmed cell death, maturation, and senescence [12]. NO also stimulates signal transduction pathways through protein kinases, cytosolic calcium (Ca2?) mobilization, and protein modification to induce resistance during plant-pathogen interactions [13]. The role of NO in the response against environmental stresses such as salinity, water stress, extreme heat and cold, mechanical wounding, UV radiation, and ozone has been reviewed previously [14].

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The role of Hb as an NO scavenger has been widely studied in several organisms. In bacteria, flavoHbs consume NO enzymatically [15, 16], in a manner equivalent to that of molecular oxygen, catalyzing the reaction of nitroxyl (HNO) [17]. Arabidopsis nonsymbiotic Hb (AHb1) scavenges NO by producing S-nitroso Hb and reduces NO emissions under conditions of hypoxic stress, indicating that it plays a role in NO detoxification [18]. In addition, the role of trHb in NO detoxification was reported by Ouellet et al. [19]. Disruption of Mycobacterium bovis bacillus glbN causes a dramatic reduction in the NO-consuming activity of stationary-phase cells. The trHbN gene detoxifies NO 20-fold more rapidly than myoglobin. Milani et al. [8] reported that Mycobacterium tuberculosis trHbN supports efficient NO dioxygenation, yielding nitrate that provides a defense barrier against nitrosative stress produced by host macrophages during lung infection. NO biosynthesis increases under salinity in many species. For instance, the endogenous level of NO was increased in olives [20] and Arabidopsis [21, 22] under salt stress. NO regulates the antioxidant defense mechanism for scavenging ROS upon exposure to salt stress [23, 24]. Herein, we provide the first evidence that trHb functions as an NO scavenger in plants. We examined the role of wheat truncated-hemoglobin (TatrHb) in NO scavenging in transgenic Arabidopsis plants by investigating phenotypes and assessing responses to salt stress. Endogenous NO emissions in transgenic plants because of salt stress were compared to those in the wild type to verify the role of TatrHb as an NO scavenger. Furthermore, the effect of insufficient NO on salt-stress sensitivity in transgenic plants was evaluated to determine whether TatrHb functions as an NO scavenger.

Materials and methods Isolation of the TatrHb gene The common wheat cultivar Geumgangmill (Accession no. IT213100), developed by the National Institute of Crop Science (RDA, Suwon, Republic of Korea), was used for isolation of the TatrHb gene. A previous study had identified the TatrHb gene that was used in our study [25]. Briefly, we collected expressed sequence tag (EST) sequences to isolate TatrHb by using ‘‘2-on-2 Hb Triticum aestivum’’ at the National Center for Biotechnology Information database (http://www.ncbi. nlm.nih.gov/). ClustalW was used in the European Bioinformatics Institute (EBI) database (http://www.ebi.ac.uk/clus talw/) to perform multiple alignments of the EST sequences. Two pairs of forward and reverse primers were designed by comparison with the EST sequences as follows: TatrHb F1 (50 AGCAGCAGGAACGATGCAGT-30 )/TatrHb R1 (50 -

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AGGAATATGGGAGCTGAAAC-30 ) and TatrHb F2 (50 ATGCAGTCGCTGCAGGACAAG-30 )/TatrHb R2 (50 CCGTTATTCAACTGGTTAAGCTTG-30 ). These primer pairs were used for initial polymerase chain reaction (PCR) amplification of the TatrHb gene. Plant materials and transformation The ecotype of Arabidopsis thaliana used in this study was a descendant of a Columbian variety (Col-0). Plants were grown in a soil mix (sunshine:perlite:vermiculite, 2:1:1) or in a Murashige and Skoog (MS) salt medium at 23 °C, with a 16-h photoperiod. The pBI121 binary vector was used to generate the plasmid for Arabidopsis transformation. The full-length TatrHb coding region was PCR amplified using the BamHI_TatrHb (50 -GGATCCATGCAGTCGCTGCAG GAC-30 ) and SacI_TatrHb (50 -GAGCTCTTATTCAACTG GTTAAGCTTG-30 ) primers, and the PCR products were cloned into a T&A cloning vector. The T&A cloning vector containing the TatrHb ORF and pBI121 binary vectors was digested using the corresponding restriction enzyme and ligated using T4 ligase (Roche, USA). Recombinant plasmids were inserted into the Agrobacterium tumefaciens strain GV3101 via the freeze–thaw method [26]. The Arabidopsis Col-0 transformation was carried out as described by Zhang et al. [27]. Growth conditions Seed surfaces of transgenic and wild-type plants were sterilized using 95 % (v/v) ethanol for 5 min and 20 % (v/v) bleach for 10 min. After vernalization for 3 days at 4 °C, seeds were germinated and grown on 0.8 % agar plates with 0.2 9 MS salts. Plates were placed vertically at an angle of 70° to allow root growth along the agar surface with a photoperiod of 16 h of light and 8 h of dark at 23 °C. The primary root numbers of transgenic and wild-type plants were determined using a stereomicroscope (Leica EZ4D, Leica Microsystems Ltd., Switzerland), and the primary root lengths of transgenic and the wildtype plants were measured using a ruler. All images were captured using a Canon Powershot SX 10IS digital color camera. Salt stress treatment Three-week-old seedlings were subjected to a salt stress treatment (i.e., 100 mM NaCl for 24 h). Samples of Arabidopsis seedlings for gene expression analysis were harvested and immediately frozen in liquid nitrogen and stored at -80 °C. Samples for NO detection by 4,5-diaminofluorescein diacetate (DAF) fluorescence were immediately transferred to the experimental loading buffer. Transgenic and wild-type plants were treated using 100 mM NaCl in MS plates for 24 and 48 h before

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detecting chlorophyll fluorescence. Seeds of transgenic and wild-type plants were germinated on 0.8 % agar plates and treated using 0.29 MS salts containing 100 mM NaCl to analyze the rate of seed germination. Subsequently, approximately 50 seeds from transgenic and the wild-type plants were scored daily for 7 days.

Measurements of K? and Na? with inductively coupled plasma (ICP) To measure the relative changes in K? and Na? levels, we analyzed K? and Na? concentrations in the wild-type and transgenic plants in response to NaCl treatment by ICP analysis. Leaves of Arabidopsis were washed in 0.2 mM CaSO4 for 5 min and then surface dried using ash-free filter paper. The fresh weight was measured before drying the samples for 24 h at 80 °C. An aliquot of dry material was digested in HNO3 for 1 h at 80 °C. After digestion, the acid concentration was diluted using double deionized water. Mineral element concentrations in the solutions were measured by ICP-OES (Perkin Elmer Optima 2000).

RNA extraction and gene expression analysis Total RNA was isolated using the TRIzolÒ reagent (Invitrogen, USA) according to the manufacturer’s instructions. The first cDNA strand was synthesized from approximately 1 lg of total RNA by using a DyNamoTM cDNA Synthesis Kit (Finnzymes, Finland) according to the manufacturer’s instructions. Semi-quantitative reverse transcription (RT) PCR was performed to assess changes in the TatrHb expression pattern between the wild-type and transgenic plants. The nucleotide sequences of the primers and the PCR conditions used in this study were as follows: TatrHb-FWD (50 -CGCGATCCAGAACCAGTACGAGTT-30 ) and TatrHb-REV (50 -TTGGGTTTGCCTTGTCATCTCATT-30 ) for TatrHb amplification; ATactin-FWD (50 -GTGCTCGAC TCTGGAGATGGTGTG-30 ) and Atactin-REV (50 -CGGCG ATTCCAGGGAACATTGTGG-30 ) for Atactin amplification; and Atrd29a-FWD (50 -CCCGGATCCTTTTCTGATA TGGTT-30 ) and Atrd29a-REV (50 -GCCCTCGAGCCGAA CAATTTATTA-30 ) for Atrd29a amplification. The cycling conditions for the TatrHb, Atactin (At2g37620), and AtRD29a (AT5g52310) amplifications were as follows: 1 min at 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 58 °C, 1 min at 72 °C, and a final extension cycle of 72 °C for 4 min. The amplified products were separated on 1 % (w/v) agarose in 19 Tris–acetate–EDTA buffer and stained using ethidium bromide. Each sample was normalized against the constitutive actin transcript level, and induction levels were compared and recorded against the transcript levels by

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densitometry using an analytical scanning system (AlphaImager 1220). Detection of endogenous NO The amounts of NO in the roots and leaf cells were measured using the fluorescent NO indicator dye, 4,5-diaminofluorescein diacetate (DAF-2 DA) (Sigma, St. Louis, MO, USA). Ten-day-old non-treated transgenic and wildtype seedlings and 2-week-old treated (100 mM NaCl) transgenic and wild-type plants were incubated using 15 lM DAF-2 DA in a loading buffer (5 mM MES-KOH, pH 5.7, 0.25 mM KCl, 1 mM CaCl2) for 15 min, which was followed by a 20-min wash in the loading buffer. All procedures for the DAF-2DA treatment were conducted in darkness. Fluorescent signals were detected using a fluorescence stereomicroscope and an in vitro multispectral imaging system (M165 FC, NuanceFX, Leica Microsystems Ltd.). The dye was excited at 488 nm, and images were captured at emission wavelengths of 515–560 nm. Imaging of chlorophyll fluorescence parameters A special version of an Imaging-PAM Chlorophyll Fluorometer (Walz, Germany) was used to investigate the spatiotemporal changes in photosynthetic parameters [28]. In this measuring system, the same array of 12 blue light-emitting diodes (peak wavelength, 450 nm; short-pass filter, 470 nm) was used for fluorescence excitation, actinic illumination, and saturating light pulses. A charge coupled device camera was equipped with a Cosmicar/Pentax lens (F1.2, f = 12 mm) with a resolution of 640 9 480 pixels. Measured light pulses equivalent to an integrated intensity of 0.4 lmol quanta m-2 s-1 were applied at low frequency (1 Hz) to measure fluorescence yield (Fo) images (quasi-dark state). Two images were measured using each pulse, one shortly before the pulse and one during the light pulse. These images were then subtracted from one another, pixel by pixel, resulting in an image that was corrected for ambient background light. In the absence of actinic illumination, the dark-level Fo and maximum fluorescence (Fm) yield were determined in conjunction with the application of a saturation pulse from which the maximum PSII quantum yield (Fv/Fm) was automatically calculated using the ImagingWin software.

Results Comparing TatrHb with other HbN-type truncated hemoglobins The sequence of TatrHb was compared to that of other mycobacteria HbN-type trHbs. The structural sequence alignment of TatrHb with other mycobacteria HbNs

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Fig. 1 Structure-based multiple sequence alignment of TatrHb with other mycobacterial HbNtype trHbs; the globin fold topological positions are shown on the top of the aligned sequences; important residues with respect to coordination of the heme and ligand-binding properties are marked and conserved residues of mycobacterial trHb families are highlighted in the black box; Mtb Mycobacterium tuberculosis, Mb M. bovis, Mm M. marinum, Ma M. avium, Ms Mycobacterium sp., TatrHb Triticum aestivum truncated hemoglobin, OstrHb Oryza sativa truncated hemoglobin, AttrHb Arabidopsis thaliana

indicated that it is approximately 35 peptides longer in size than that of its counterpart in the C-terminal and N-terminal sequences. TatrHb contained several specific residues of bacterial HbN, such as E15, E7, CD1, B10, and F8 (Fig. 1). trHbN functions alongside oxide dioxygenase (NOD) to relieve nitrosative stress in its host. Recently, Oliveira et al. [29] reported that the E15 residue is involved in ligand-induced conformational changes for NO detoxification by modulating the diffusion of NO. E7 residues are common in trHb and may be instrumental for ligand entry into the heme distal site [30]. The CD1 residue is in the middle of the C helix and plays a role in binding to the heme, the B10 residue is located in the H-bonding side chains within the distal heme pocket, and F8 is a hemeproximal residue of trHb. CD1, B10, and F8 residues of trHb possess ligand-binding properties and are important for heme formation [6]. Although the sequence homology of plant trHbs showed much higher similarity than that of other mycobacteria, the majority of the globin fold topological positions and important residues, with respect to coordination of the heme and ligand-binding properties, were detected in plants and mycobacteria trHbs. Effect of salt stress on the Na? to K? ratio in transgenic plants Kim et al. [25] reported that the transcripts of TatrHb showed an immediate response to exogenous NO (sodium nitroprusside, NO donor) and NaCl treatment. Therefore, we developed transgenic Arabidopsis plants to reveal the

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role of trHb in NO scavenging, similar to the method for understanding the role of HbN-type trHbs in mycobacteria. Arabidopsis T3 plants overexpressing TatrHb have very short roots when compared to those of the wild type. The primary root lengths of transgenic plants were 62 and 78 % of that of the wild type, respectively. In addition, the average number of lateral roots for the control plants was 7.7; the average number of lateral roots of the transgenic plants was reduced to 4.1 and 5.0, respectively (Fig. 2). Transgenic Arabidopsis plants were grown in 100 mM NaCl for 24 h, and expression of the salt-stress-responsive gene rd29a in Arabidopsis was performed simultaneously with identical cDNA to verify the effect of the treatment (Fig. 3a). The expression level of Atrd29a dramatically increased 6.1-fold at 24 h after the salt treatment. In addition, the constitutive expression of TatrHb was detected in transgenic plants under the salt stress condition, and there were no changes in the expression level between the normal and salt stress conditions. We measured K?, Na?, and the Na? to K? ratio to investigate the effects of NaCl on wild-type and transgenic plants. The Na? concentration increased in both wild-type and transgenic plants in accordance with an increase in NaCl concentration, whereas the K? concentration decreased in both the wildtype and transgenic plants. However, the degree of increase in Na? and decrease in K? for transgenic plants was greater than that for the wild-type plants (Fig. 3b, c). The changes in Na? and K? concentrations led to a greater increase in the Na? to K? ratio in transgenic plants when compared to that in the wild-type plants (Fig. 3d).

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Fig. 2 Comparison of root development in overexpressed TatrHb transgenic Arabidopsis and wild-type plants at age 3 weeks: a wild type, b, c transgenic plants, d mean and standard error of the primary root length, e means of the lateral roots of 30 individual 3-week-old wild-type and transgenic plants. The error bars indicate the SD; *, **Significant differences in comparison with the wild type at the 0.01 \ P \ 0.05 and P \ 0.01 levels, respectively, using the Student’s t test

Fig. 3 a Transcript accumulation profile of TatrHb transgenic plants and the saltstress-responsive gene rd29a in Arabidopsis under salt stress to verify the effect of the treatment; effect of NaCl on K? (b) and Na? concentrations (c), and the Na?/K? ratio (d) in the wild-type and TatrHb transgenic plants under salt stress; wild-type and transgenic plants were exposed to concentrations of NaCl (0, 50, 100, and 200 mM) for 48 h; the contents of K? and Na? in wildtype and transgenic plants were determined using ICP; the means and standard errors of three independent experiments are shown

Confocal microscopic observation of roots under salt stress To determine if a greater change in the Na? to K? ratio in transgenic plants might be caused by constitutively expressed TatrHb, we evaluated the endogenous NO levels in the roots of transgenic and wild-type plants under salt stress treatment, an NO donor [sodium nitroprusside (SNP)], and an NO scavenger (c-PTIO) by using DAF fluorescence (Fig. 4). The endogenous NO level of

transgenic plants was 65 % of the wild type in the normal condition (Fig. 4a, e, i), and the endogenous NO level of the wild type increased to 122 and 134 % by the NaCl and SNP treatments, respectively (Fig. 4c, d, i). Furthermore, the endogenous NO level of the wild type was reduced to 70 % by c-PTIO treatment (Fig. 4b, i). In transgenic plants, the endogenous NO level was slightly reduced to 85 % by c-PTIO and increased to 110 % by NaCl (Fig. 4f, g, i). Furthermore, in transgenic plants, the endogenous NO level was increased to 157 % by the SNP treatment when

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Fig. 4 Effects of NaCl, c-PTIO, and SNP on endogenous NO levels in the roots of wild-type and transgenic plants; NO levels were detected by confocal microscopy in wild-type roots stained with DAF-2DA (a), treated for 2 h with 100 mM c-PTIO (NO scavenger) (b), 100 mM NaCl (c), and 0.1 mM SNP (NO donor) (d); e– h representative NO levels in the roots of transgenic plants treated with the control, 100 mM c-PTIO, 100 mM NaCl, and 0.1 mM SNP for 2 h; mean relative DAF-2DA fluorescence densities for the roots of wild-type and transgenic plants corresponding to a–h are given in i; the means of seven independent Arabidopsis root experiments are shown. The error bars indicate the SD; *, **Significant differences in comparison with the wild type at the 0.01 \ P \ 0.05 and P \ 0.01 levels, respectively, using the Student’s t test

compared to that in the normal condition (Fig. 4h, i). The rates of change in endogenous NO levels were more notable in the wild type than in the transgenic plants when both plant types were treated using SNP and NaCl, which are known to induce NO. By using the above results, we measured the effects of decreased endogenous NO by TatrHb in transgenic plants. Effect of salt stress on plants overexpressing TatrHb Changes in the photosynthetic activity were analyzed using an Imaging-PAM Chlorophyll Fluorometer to investigate the effect of TatrHb on the tolerance of transgenic and wild-type plants to salt stress. The maximum photosystem (PS) II quantum yield (Fv/Fm) was measured after 100 mM NaCl treatment (Fig. 5). The Fv/Fm of PS II was [0.7 in intact leaves of transgenic and wild-type plants. The Fv/Fm of transgenic plants was significantly lower than that of the wild-type plants at 24 h after treatment, and the difference between transgenic and wild-type plants markedly increased at 48 h. The low value of Fv/Fm (0.428) in transgenic plants at 48 h after treatment indicates that the transgenic plants suffered more severe damage because of salt stress than the wild-type plants (Fig. 5).

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Seeds were germinated in the presence of stress, and the primary root length and Fv/Fm were measured to elucidate the effect of TatrHb on seed germination under salt stress. The Fv/Fm of Arabidopsis plants germinated in a saltcontaining medium was measured to evaluate the effect of salt stress on the germination of transgenic and wild-type plants (Fig. 6). Interestingly, the Fv/Fm value of the leaves of salt-treated wild-type plants were the same as that of the intact leaves ([0.7), even though they had shorter root lengths and fewer leaves than those in the non-treated wild type (Fig. 6). The Fv/Fm of the leaves of salt-treated transgenic plants decreased to 0.56, indicating that germination of transgenic seeds was more sensitive to NaCl than that by the wild-type seeds. Treatment with NO or reactive oxygen donors such as SNP can enhance seed germination [10, 31]. To verify the effect of inhibiting seed germination by TatrHb in transgenic plants, which has a putative NO scavenging role, we measured the effect of NaCl on the germination rates of wild-type and transgenic seeds. The gap in the germination rate for wild-type and transgenic seeds under normal conditions was 2–6 % from 2 to 7 days after germination (DAG), whereas it was 32 % on 2 DAG and 31 % on 3 DAG under the salt stress condition. The final germination rates of the wild-type and transgenic

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Fig. 5 Comparison of the effects of salt stress on chlorophyll fluorescence parameters: a wild type and b transgenic plants; Arabidopsis were treated with 100 mM NaCl and analyzed after 24 and 48 h; images of the maximum photosystem (PS) II quantum yield, Fv/Fm, are shown; the color code depicted at the bottom of each image ranges from 0.000 (black) to 1.000 (purple); c quantitative analysis of the time-dependent changes induced by 100 mM NaCl treatment on maximum PS II quantum yield, Fv/Fm; Arabidopsis plants were treated with 100 mM NaCl and analyzed after 24 and 48 h; the means of three independent Arabidopsis leaf experiments are shown. The error bars indicate the SD; *, **Significant differences in comparison with the wild type at the 0.01 \ P \ 0.05 and P \ 0.01 levels, respectively, using the Student’s t test. (Color figure online)

seeds under salt stress were 76 and 62 %, respectively. The results indicate that the negative effect on seed germination was more severe in transgenic plants than wild-type plants under the NaCl treatment.

Discussion NO is a bioactive molecule that functions in numerous physiological processes in plants. In addition, there have been significant studies investigating the role of NO involvement in root formation. The NO donor SNP induces growth elongation in maize root segments [32]. NO mediates the auxin response leading to adventitious root formation in the cucumber plant [33] and promotes lateral root development, an auxin-dependent process. NO and cGMP are involved in controlling cell elongation and root extension in Arabidopsis [34]. The role of trHbs in NO detoxification was previously studied and identified in bacteria. The trHbN from M. tuberculosis actively detoxified NO and protected aerobic respiration from NO inhibition [19]. Lack of trHbN in M. bovis resulted in a decrease in respiration activity upon exposure to NO [35]. NO generated by the addition of an

NO donor caused a tenfold increase in gene expression of HbN-type trHbs in Frankia [36]. In the current study, transgenic plants overexpressing TatrHb had shorter root lengths than those of the wild type (Fig. 1), suggesting that overexpressed TatrHb continuously reacts with NO in transgenic plants, and this constant reaction, which converts NO to a nitrate anion (NO-3) or directly binds to NO, may lead to a lack of endogenous NO in transgenic plants. The deficiency of endogenous NO in the roots of transgenic plants did not sufficiently promote root elongation. This result corresponded with the observation that the amount of endogenous NO detected by DAF florescence was much higher in the wild-type plants than that in the transgenic plants. NO was scarcely detected in transgenic plants, whereas it was widely distributed in the wild-type plants (Fig. 2). These results indicate that TatrHb plays a crucial role in scavenging NO, which ultimately inhibits root elongation. NO has been suggested to be involved in responses to drought stress, heat stress, disease resistance, and apoptosis [10, 37–41]. It has been shown that the exogenous application of NO donors could enhance salinity tolerance in many species of plants, including reed (Phragmites communis), sunflower (Lupinus luteus), wheat, rice, bitter

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Fig. 6 Comparison of the effects of germination on chlorophyll florescence parameters in plants under salt stress: a wild type and b transgenic plants; Arabidopsis seeds were germinated on Murashige and Skoog (MS) media with 100 mM NaCl and analyzed 10 days after germination; images of the maximum photosystem (PS) II quantum yield, Fv/Fm, are shown; the color code depicted at the bottom of each image ranges from 0.000 (black) to 1.000 (purple); c quantitative analysis of the effects of 100 mM NaCl treatment on

germination as assessed by the maximum PS II quantum yield, Fv/ Fm; Arabidopsis seeds were germinated on MS media with 100 mM of NaCl and analyzed 10 days after germination; the means of three independent Arabidopsis leaf experiments are shown. The error bars indicate the SD; *, **Significant differences in comparison with the wild type at the 0.01 \ P \ 0.05 and P \ 0.01 levels, respectively, using the Student’s t test. (Color figure online)

orange (Citrus aurantium L.), and tobacco-cell suspensions [14]. Zang et al. [36] previously reported that NO acts as a signal molecule in the NaCl treatment by increasing the activities of vacuolar H?-ATPase and H?-PPase to facilitate Na?/H? exchange. In addition, NO synthase (NOS)dependent nitric oxide production was associated with salt tolerance in Arabidopsis. The accumulation of Na? in Arabidopsis mutant (Atnoa1) plants increased more than in the wild type under NaCl treatment; furthermore, Atnoa1 was more sensitive to seed germination when exposed to the NaCl treatment than the wild type [21]. Moreover, Qiao et al. [22] reported that Atnoa1 plants rescued by the introduction of OsNOA1 recovered seedling growth, vegetative growth under normal condition, and the salt tolerance Atnoa1 plant. Treatment by using 100 lM SNP with 400 mM NaCl significantly increased the density of salt crystals and the salt-secretion rate of leaves; it also maintained a low Na? to K? ratio in Avicennia marina [42]. We hypothesized that endogenous NO in transgenic plants under salt stress might be reduced by constant TatrHb expression, which can act as an NO scavenger; therefore, we monitored endogenous

NO in transgenic and wild-type plants under salt stress to examine changes in the NO concentration. NO increased significantly in the leaves and roots when the wild-type plants were exposed to NaCl (Fig. 4). The increased endogenous NO level in response to salt stress induced by NaCl resulted in salt tolerance, to some extent, in the wildtype plants (Fig. 6). Zhang et al. [43] previously reported that NaCl induces a transient increase in the NO level in maize leaves over a 6-h period. SNP and NaCl treatments stimulated vacuolar H?-ATPase and H?-PPase activity, resulting in increased H?-translocation and Na?/H? exchange. The TatrHb transcription level in transgenic plants increased 2.4-fold under salt stress (Fig. 3). This result suggests that the transient increase in the NO level induced by salt stress caused an upregulation of TatrHb in transgenic plants. The upregulated TatrHb, in turn, may play a role in NO detoxification and NO dioxygenation, similar to that by bacterial trHbs [8, 19]. In accordance with these findings, the presence of NO, as measured by the intensity of green fluorescence, was only rarely detected in the leaves and roots of transgenic plants under salt stress

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(Fig. 4). Additionally, the Fv/Fm of transgenic plants dropped drastically when compared to that of the wild-type plants (Fig. 5). The observation that transgenic plants, in which NO was rarely detected in both the roots and leaves, were severely damaged by salt stress when compared to the wild-type plants provides support for the role of TatrHb as an NO scavenger. Recently, NO and S-nitrosylation have emerged as potential new paradigms in signal transduction and regulation for protein function. NO and protein S-nitrosylation are important mediators in the process of H2O2-induced leaf cell death in rice [44]. NO enhances desiccation tolerance of recalcitrant Antiaris toxicaria seeds via protein Snitrosylation and carbonylation [45]. Moreover, posttranslational modifications such as S-nitrosylation and denitrosylation modulated respiratory and photorespiratory pathways under salt stress in pea plants [46]. Tanou et al. [47] reported that both H2O2 and SNP pretreatments before salinity stress alleviated salinity-induced protein carbonylation and led to a decrease in the levels of accumulated Snitrosylated proteins in the leaves when compared to those in unstressed control plants. In this study, vernalized seeds were germinated on MS media containing 100 mM NaCl to compare the effect of salt stress on germination between transgenic and wild-type plants. The Fv/Fm of transgenic plants was also low (0.56) in the salt-containing media (Fig. 6). The Fv/Fm of the wild-type plants germinated on salt-containing media was not greatly affected by salt stress when compared to that of the transgenic plants (Fig. 7). These results suggest that the signal cascade of salt stress, including NO, NADPH, H2O2, H?-ATPase, and S-nitrosylation, in the wild type could be continued, with the normal production of endogenous NO in vivo [48]. Thus, Fv/Fm was less affected by salt stress in the transgenic plants, even though they had fewer leaves and shorter roots than those of the non-treated wild-type plants. In contrast, because endogenous NO was continuously consumed by overexpressed TatrHb in transgenic plants, NO was not available in sufficient amounts to stimulate the salt tolerance signal transduction pathway. Removing NO produced an abnormal defense mechanism in the transgenic plants. As a result, in plants overexpressing TatrHb under salt stress, the activity of the plasma membrane H?-ATPase could not be regulated by H2O2 or NO, post-translational modification decreased, and Na? accumulation increased. These processes resulted in sensitivity to salt stress in transgenic plants because of insufficient NO. In the current study, TatrHb overexpressed Arabidopsis plants showed lower endogenous NO levels and higher sensitivity to salt stress than those of the wild-type plants. These findings indicate that reduced endogenous NO by TatrHb is closely related to sensitivity under salt stress in Arabidopsis. Furthermore, the results support the hypothesis

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Fig. 7 Effect of NaCl on the germination rate of transgenic and wildtype seeds; the rate of seed germination on 0.2 9 Murashige and Skoog (MS) salt media containing 100 mM NaCl for transgenic plants (white circle) and the wild type (white square), and without 100 mM NaCl for transgenic plants (black circle) and the wild type (black squares)

that plant trHb shares certain NO scavenging functions and characteristics with bacterial trHb. Acknowledgments This study was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ008031), Rural Development Administration, Republic of Korea and supported by a Korea University Grant. This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIP) (No. 2012M2A2A6035566).

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