Skip to main content
NCBI home page
Journal of Experimental Botany logoLink to Journal of Experimental Botany
. 2013 Feb 1;64(5):1367–1379. doi: 10.1093/jxb/ers400

Manipulation of arginase expression modulates abiotic stress tolerance in Arabidopsis: effect on arginine metabolism and ROS accumulation

Haitao Shi 1, Tiantian Ye 1,2, Fangfang Chen 1, Zhangmin Cheng 1,2, Yanping Wang 1, Pingfang Yang 1, Yansheng Zhang 1, Zhulong Chan 1,*
PMCID: PMC3598423  PMID: 23378380

Abstract

Arginine is an important medium for the transport and storage of nitrogen, and arginase (also known as arginine amidohydrolase, ARGAH) is responsible for catalyse of arginine into ornithine and urea in plants. In this study, the impact of AtARGAHs on abiotic stress response was investigated by manipulating AtARGAHs expression. In the knockout mutants of AtARGAHs, enhanced tolerances were observed to multiple abiotic stresses including water deficit, salt, and freezing stresses, while AtARGAH1- and AtARGAH2-overexpressing lines exhibited reduced abiotic stress tolerances compared to the wild type. Consistently, the enhanced tolerances were confirmed by the changes of physiological parameters including electrolyte leakage, water loss rate, stomatal aperture, and survival rate. Interestingly, the direct downstream products of arginine catabolism including polyamines and nitric oxide (NO) concentrations significantly increased in the AtARGAHs-knockout lines, but decreased in overexpressing lines under control conditions. Additionally, the AtARGAHs-overexpressing and -knockout lines displayed significantly reduced relative arginine (% of total free amino acids) relative to the wild type. Similarly, reactive oxygen species accumulation was remarkably regulated by AtARGAHs under abiotic stress conditions, as shown from hydrogen peroxide (H2O2), superoxide radical (Inline graphic) concentrations, and antioxidant enzyme activities. Taken together, this is the first report, as far as is known, to provide evidence that AtARGAHs negatively regulate many abiotic stress tolerances, at least partially, attribute to their roles in modulating arginine metabolism and reactive oxygen species accumulation. Biotechnological strategy based on manipulation of AtARGAHs expression will be valuable for future crop breeding.

Key words: Abiotic stress, arginase, arginine metabolism, nitric oxide, polyamines, reactive oxygen species.

Introduction

As one of the most expensive nutrients to supply, nitrogen has crucial roles in plant growth, development, and production (Brauc et al., 2012; Ma et al., 2012). Arginine is an important amino acid in plants as a medium for the transport and storage of nitrogen and a precursor for the synthesis of other amino acids and polyamines (Flores et al., 2008; Brauc et al., 2012). Three pathways have been shown to be responsible for the metabolism of arginine in higher plants: (1) via arginine to ornithine and urea by arginase (also known as arginine amidohydrolase, ARGAH); (2) via arginine decarboxylase to agmatine and then, via agmatine iminohdrolase and N-carbamoylputrescine amidohydrolase, to putrescine; and (3) via arginine to nitric oxide (NO) and citrulline by NO synthase (Flores et al., 2008; Gupta et al., 2011; Brauc et al., 2012). The reaction products may be further metabolized to other amino acids and polyamines, including proline, putrescine, spermidine, and spermine, all of which play crucial roles in wide developmental processes and in biotic and abiotic stress responses (Flores et al., 2008; Wang et al., 2011b; Brauc et al., 2012; Shi et al., 2012a,b). Polyamines (mainly putrescine, spermidine, and spermine) are scavengers of reactive oxygen species (ROS), and proline is one of the most common compatible metabolites to regulate cell-membrane stability and balance osmotic pressure under various stress conditions (Wimalasekera et al., 2011; Brauc et al., 2012).

As an important route for arginine metabolism, arginase enzyme activity has been described in many plant species, and arginase gene expression can be enhanced by wounding, methyl jasmonate, and the phytotoxin coronatime (Chen et al., 2004). AtARGAH1 (At4g08900) and AtARGAH2 (At4g08870) have been cloned and identified in Arabidopsis. T-DNA insertion mutants of AtARGAHs result in increased NO accumulation and the propensity to form lateral and adventitious roots under the control of auxin signalling (Krumpelman et al., 1995; Flores et al., 2008). Furthermore, only the expression of AtARGAH2 could be induced by methyl jasmonate and Botrytis cinerea (Brownfield et al., 2008; Brauc et al., 2012), and overexpression of AtARGAH2 in Arabidopsis confers increased tolerance to B. cinerea (Brauc et al., 2012). Additionally, AtARGAH2 was found to be largely induced in auxin/cytokinin-triggered cellular proliferation, and enhanced callus development was observed in an argah2 mutant during clubroot infection (Gravot et al., 2012). Recently, Ma et al. (2012) found that the arginase encoded by OsARG is not only a key enzyme in arginine catabolism, but also that it plays critical roles in panicle development and grain production in rice, especially under insufficient exogenous nitrogen conditions.

To date, the in vivo role of AtARGAHs in plant tolerance to abiotic stress remains largely unknown. The present research investigated the influence of manipulation of AtARGAHs expression on arginine metabolism and ROS accumulation, as well as plant responses to abiotic stress including water deficit, salt, and freezing. How manipulation of arginase expression influences abiotic stress tolerance through arginine metabolism or ROS accumulation is also discussed.

Materials and methods

Plant materials and growth conditions

Arabidopsis thaliana ecotype Columbia was used in this study. The mutants of argah1 (SALK_057987) and argah2 (SAIL_181_C11) were obtained from the Arabidopsis Biological Resource Center as described previously (Flores et al., 2008). The argah1argah2 double mutant was subsequently generated by crossing argah1 and argah2. Arabidopsis seeds were sown in plastic containers filled with soil or on Murashige and Skoog (MS) medium containing 1% sucrose (w/v) in the growth chamber after stratification at 4 °C for 3 days in darkness. The growth chamber was controlled at an irradiance of about 120–160 µmol quanta m–2 s–1 at 22–25 °C with 65% relative humidity under a 16/8 light/dark cycle. Irrigated nutrient solution was given twice a week.

To characterize the effects of abiotic stress on AtARGAHs expression, 21-day-old seedlings grown on MS medium were transferred to fresh MS liquid medium supplemented with water (control), 30 µM abscisic acid (ABA), and 300mM NaCl, or were subjected to dehydration and 4 °C stress treatments for 1 and 3h, respectively.

Plant transformation and selection

To obtain the overexpressing transgenic lines, the full-length cDNA fragments of AtARGAH1 and AtARGAH2 were inserted into the modified pEGAD vector under the control of CaMV 35S promoter (Cutler et al., 2000). The corresponding specific primers of the above constructions are listed in Supplementary Table S1 (available at JXB online). Transgenic plants were selected with BASTA resistance and were confirmed by PCR as previously described (Shi et al., 2012b).

Semi-quantitative reverse-transcription PCR and quantitative real-time PCR

Total RNA was isolated from 200mg leaves using TRIzol reagent (Invitrogen) and treated by RQ1 RNase-free DNase (Promega) to remove genomic DNA. First-strand cDNA was synthesized using reverse transcriptase (TOYOBO) from 2 µg total RNA, and 1 µl cDNA was added to 30 µl PCR mixture for semi-quantitative reverse-transcription PCR (RT-PCR). For quantitative real-time PCR, a CFX96 Real Time System (BIO-RAD) with SYBR-green fluorescence and the comparative ΔΔCT method was used as previously described (Shi et al., 2012b). All experiments were repeated at least three times and the expression levels were standardized with UBQ10. The specific primers of analysed genes are listed in Supplementary Table S1.

Determination of arginine concentration and total free amino acids

For amino acid analysis, 0.3g 2-week-old plant leaves were ground and resuspended in 1ml water as previously described (He et al., 2004). After centrifugation at 15,294 g for 15min, the supernatant was filtered with a 0.22 µm nylon filter. Arginine concentration was quantified by the formation of stable yellow species on the addition of thymol-sodium hypobromite reagent under alkaline conditions (Sastry and Tummuru, 1984). Total free amino acids were measured using Cd-nihydrin method as described previously (Fisher et al., 2001).

Determination of endogenous NO and NO synthase activity

The endogenous NO content of Arabidopsis leaves was analysed by labelling with the NO-sensitive fluorescence dye 3-amino,4-aminomethyl-2’,7’-difluorescein diacetate (DAF-FM-DA) and quantified by the haemoglobin assay as described previously (Shi et al., 2012b). The fluorescence image of plant leaves labelled with NO was taken from fluorescence stereomicroscope (DP72, Olympus), and the NO content was quantified using a fluorometer (Molecular Device Company, Spectra Max M2). All fluorescences were measured at excitation of 485nm and emission of 510nm. For the haemoglobin assay, the NO content in leave extracts was quantified spectrophotometrically by measuring the conversion of oxyhaemoglobin to methaemoglobin as previously described (Murphy and Noack, 1994). NO synthase activity was determined by adding assay mixture (NADPH, l-arginine, Ca2+-CaM, BH4, FAD, FMN, oxygen) in the presence and absence of excess NO synthase inhibitor as described previously (Shi et al., 2012b).

Quantification of polyamines

Polyamines in 2-week-old plant leaves were extracted and quantified by HPLC as previously described (Walter and Geuns, 1987) with minor modifications. Briefly, the column (length 15cm, internal diameter 4.6mm) was filled with 5 µm reversed-phase material (HeSep C18-T, Weltech, Wuhan) and heated at 45 °C for analysis. The solvent was acetonitrile:H2O (72:28) with flow 1.2ml/min and the injection volume was 30 µl.

Assessment of salt, water deficit, and freezing tolerance

Wild-type, AtARGAHs-overexpressing lines, and argah mutant plants were grown in the growth chamber in soil for 2 weeks. For water deficit stress treatment, 2-week-old plants were subjected to water deficit condition by withholding water for 18 d and then rewatered for 10 d. For salt stress treatment, the NaCl concentration was increased stepwise by 50mM every 2 d to the indicated concentration for the next 4 weeks, as previously described (Chan et al., 2011). The whole-plant freezing test was analysed as described by Verslues et al. (2006) with some modifications. Briefly, 2-week-old seedlings were cold acclimated at 4 °C in the light for 1 week, then the plants were placed in a growth chamber set to –1 °C for 16h. Ice chips were sprinkled on these plants before the chamber was programmed to cool at 1 °C per hour until –6 °C was reached. Plants were removed after being frozen, thawed at 4 °C for 12h in the dark, and then transferred to a growth chamber at 22 °C. The survival rate was scored 7 d later.

Measurement of electrolyte leakage, lipid peroxidation, and leaf water status

For the determination of electrolyte leakage, about 0.1g plant leaves were incubated in 10ml deionized water, shaken on a gyratory shaker at room temperature for 6h at about 150rpm. First, the initial conductivity (Ci) was measured using a conductivity meter (Leici-DDS-307A, Shanghai, China). The samples were then boiled for 20min to kill the leaf tissues and completely induce all electrolytes of the solution. After cooling to room temperature, the conductivity of the killed tissues (Cmax) was determined. The relative electrolyte leakage could be calculated as (Ci/Cmax) × 100%.

As major indicator of lipid peroxidation, malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) method as previously described (Shi et al., 2012b). Briefly, 1g plant leaves were incubated in 5ml TBA reagent (0.25%, w/v, TBA in 10%, w/v, trichloroacetic acid), and then heated at 100 °C for 20min. After cooling to room temperature, the MDA content of sample was calculated by subtracting the non-specific absorption at 600nm from the absorbance of the sample supernatant at 532nm.

For water loss rate assay, the detached leaves were quantified initial fresh weight (FW) and placed into growth chamber. The FW was recorded every 30min for 8h and water loss rate was calculated from the relative decrease in FW. For stomatal aperture assay, plant leaves were incubated in stomatal opening buffer (5mM MES, 10mM KCl, 50mM CaCl2, pH 6.15) for 2h. After treatment, stomatal aperture width was measured in stereomicroscope (DP72, Olympus) equipped with a CCD camera, and at least 100 stomata were tested for each line.

Determination of ROS accumulation and assay of antioxidant enzyme activities

Hydrogen peroxide (H2O2) and superoxide radicals (Inline graphic) were stained using diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively, according to Wang et al. (2011b). For H2O2 detection, the seedlings were immersed in NBT solution (1mg ml–1) in 10mM phosphate buffer (pH 7.8) at room temperature for 8h. For localization of v, the Arabidopsis seedlings were incubated in DAB solution (pH 3.8, 1mg ml–1) at room temperature for 8h. The stained seedlings were then transferred to 70% (v/v) ethanol to remove chlorophyll and visualize the blue and brown spots for H2O2 and Inline graphic, respectively. The H2O2 content was quantified as described previously (Shi et al., 2012c) to confirm the DAB staining. Briefly, 1ml plant extract using 50mM sodium phosphate buffer (pH 7.8) was mixed with 1ml of 0.1% (w/v) titanium sulphate (in 20%, v/v H2SO4) thoroughly for 10min. After centrifugation at 15,294 g for 10min, the absorbance of the supernatant was measured at 410nm. The concentration of proteins was quantified using the Bradford method (Bradford, 1976). Catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), and peroxidase (POD, EC 1.11.1.7) activities were analysed as described previously (Shi et al., 2012c).

Statistical analysis

All experiments were repeated at least three times in independent experiments, and the leaf samples of independent experiments were harvested from at least 30 seedlings per genotype. In these results, Student’s t-test was used to determine the significant difference between the wild type and the different AtARGAHs lines at P < 0.05.

Results

The expression patterns of AtARGAHs after abiotic stress treatments

First, this study examined the expression patterns of AtARGAHs in response to exogenous stress treatments including abscisic acid, dehydration, NaCl, and cold. When subjected to dehydration stress for 1 and 3h, transcript levels of AtARGAH1 and AtARGAH2 were largely activated, as well as significantly by abscisic acid, NaCl, and cold stresses at 1 and 3h after treatments (Fig. 1). The induced expression of AtARGAHs under these stress conditions suggested the role for AtARGAHs in tolerance to these stresses, especially in response to dehydration stress.

Fig. 1.

Fig. 1.

Open in a new tab

Expression levels of AtARGAHs after abiotic stress treatments by quantitative real-time PCR. The expression patterns of AtARGAH1 (A) and AtARGAH2 (B) were determined after control, ABA, dehydration, NaCl, and 4 °C treatments for 1 and 3h. The expression levels at 1h under control conditions were normalized as 1.0. Values are mean ± SD of three or more independent experiments.

Characterization of AtARGAHs-overexpressing and -knockout lines

To investigate the biological function of AtARGAHs in plants, previously described T-DNA insertion lines for argah1 and argah2 (Flores et al., 2008) were isolated in this study and an argah1argah2 double mutant was generated by crossing argah1 and argah2. There was no detectable expression of AtARGAH1 and AtARGAH2 in homozygous mutants, suggesting that argah1, argah2, and argah1argah2 are null alleles (Fig. 2A and B). To further assess the role of AtARGAHs, this study also generated AtARGAH1- and AtARGAH2-overexpressing transgenic lines and confirmed the overexpression of AtARGAH1 or AtARGAH2 at the mRNA level in these transgenic lines. All three independent overexpressing lines showed significantly higher mRNA levels compared with the wild type (Fig. 2A and B).

Fig. 2.

Fig. 2.

Open in a new tab

Effect of AtARGAHs expression changes on arginine concentration. (A and B) AtARGAHs expression in T-DNA insertion mutants and transgenic overexpressing plants by semi-quantitative RT-PCR (A) and quantitative real-time PCR (B). UBQ10 was used as a control. (C) Relative arginine (% of total free amino acids) in 2-week-old plant leaves. Values are mean ± SD of three or more independent experiments.

Effect of AtARGAHs expression on arginine concentration

Through quantification of arginine concentration, no significant differences were observed between the wild type and the AtARGAHs-knockout and -overexpressing lines when the data was expressed as µM (g FW)–1 (Supplementary Fig. S1A). However, these lines had higher concentrations of total free amino acids than the wild type (Supplementary Fig. S1B). Therefore, the relative arginine contents (% of total free amino acids) in different AtARGAHs lines were significantly lower than the wild type (Fig. 2C).

Influence of AtARGAHs expression on NO content

NO accumulation was significantly enhanced in the roots of argah1-1 and argah2-1 (Flores et al., 2008). However, NO synthase activity changes in these mutants were still unknown. Here, the results showed that argah1, argah2, and argah1argah2 mutants had significantly higher NO synthase activities than the wild type, while the AtARGAH1- and AtARGAH2-overexpressing lines exhibited relatively lower NO synthase activities (Fig. 3A). Both of the DAF-FM-DA and haemoglobin assays showed that argah1, argah2, and argah1argah2 mutants had higher levels of NO emission in the leaves, with at least 40% increased NO accumulation than the wild type (Fig. 3B and C). In contrast, the AtARGAH1- and AtARGAH2-overexpressing lines only showed about 50% NO relative to the wild type (Fig. 3B and C). These results indicated that both AtARGAH1 and AtARGAH2 could negatively regulate NO synthase activity and the intracellular NO content of plant.

Fig. 3.

Fig. 3.

Open in a new tab

Influence of AtARGAHs expression changes on NO synthase activity and NO content. (A) NO synthase activity in leaves of the wild type and the different AtARGAHs lines by the haemoglobin assay. (B) NO content in leaves of the wild type and the different AtARGAHs lines by the DAF-FM-DA assay. Bars = 50 µm. (C) NO content in leaves by the haemoglobin assay. Values for the wild type were normalized as 1.0. Values are mean ± SD of three or more independent experiments (this figure is available in colour at JXB online).

Influence of AtARGAHs expression on polyamine concentrations

Besides NO synthase activity and NO content, this study also examined the influence of AtARGAHs expression on gene expression of arginine decarboxylase pathway and polyamines. Quantitative real-time PCR results indicated that the AtARGAHs-knockout mutants exhibited much higher expression levels of AIH (agmatine iminohdrolase), NLP1 (N-carbamoylputrescine amidohydrolase), and ADC1 and ADC2 (arginine decarboxylases), whereas the AtARGAH1- and AtARGAH2-overexpressing lines had significantly lower mRNA levels when compared to the wild type (Fig. 4A). Although spermidine content showed no significant differences between AtARGAH lines and the wild type, putrescine and spermine concentrations were significantly higher in the AtARGAHs-knockout mutants, and significantly lower in the AtARGAH1- and AtARGAH2-overexpressing lines when compared to the wild type (Fig. 4B and C). These observations indicated that both AtARGAH1 and AtARGAH2 could negatively regulate polyamines through arginine decarboxylase pathway.

Fig. 4.

Fig. 4.

Open in a new tab

Effect of AtARGAHs expression changes on polyamines concentrations. (A) Real-time PCR analysis of the expressions of ADC1, ADC2, AIH, and NLP1 in 2-week-old plant leaves (three or more independent experiments). (B) Quantification of putrescine, spermidine, and spermine in 2-week-old plant leaves using HPLC. Values are mean ± SD. Seeds from different lines were sown once and HPLC analysis was performed in triplicate using different leaf materials.

Performance of AtARGAHs-overexpressing and -knockout lines under water deficit and salt stress

To dissect whether AtARGAHs could play roles in response to water deficit stress, a water deficit stress tolerance test was conducted. As typical physiological indices for estimation of stress tolerance in plants (Wang et al., 2011a), electrolyte leakage and survival rate were also determined to access plasma membrane damage and stress tolerance among different plants. After water deficit stress, about 60% of the wild-type plants died, while 70% of the AtARGAHs-overexpressing lines died with higher level of electrolyte leakage when compared with the wild type (Fig. 5A–C). As for the argah1, argah2, and argah1argah2 mutants, only 20–30% plants died under water deficit stress and the relative electrolyte leakages were remarkably lower than the wild type (Fig. 5A–C). Leaf water loss, another important parameter for evaluating plant tolerance to water deficit stress, was also examined among these lines. Consistently, all argah1, argah2, and argah1argah2 mutants had reduced water loss in comparison with the wild type, whereas the AtARGAHs-overexpressing lines lost more water than the wild-type plants (Fig. 5D). Furthermore, argah1, argah2, and argah1argah2 mutants showed significantly less stomatal aperture width than the wild type, while the AtARGAHs-overexpressing lines did not (Fig. 5E). As the open/closed status of stomata is related to water loss, so the less stomatal aperture width might explain the lower water loss rate of the mutants, and the good water status of the mutants might reasonably contribute to the enhanced tolerance.

Fig. 5.

Fig. 5.

Open in a new tab

Performance of the wild type and the different AtARGAHs lines under water deficit and salt stress conditions. (A) Two-week-old plants after 28 d under control conditions, after 10 d recovery after water deficit treatment for 18 d, and after 28 d after 150mM NaCl treatment. (B) Electrolyte leakage of plants under control, water deficit, and salt stress conditions for 12 d. (C) Survival rate of plants under control, water deficit, and salt stress conditions for 4 weeks. (D) Leaf water loss rate of the wild type and the different AtARGAHs lines at ambient environment. (E) Stomatal aperture width of leaves from the wild type and the different AtARGAHs lines. Values are mean ± SD of three or more independent experiments (this figure is available in colour at JXB online).

For salt stress treatment, 150mM NaCl was imposed on 2-week-old plants for 4 weeks. Under normal growth conditions, all ARGAHs-overexpressing and -knockout lines showed no significant difference in comparison to the wild-type plants in terms of the relative electrolyte leakage and survival rate (Fig. 5A–C). When 150mM NaCl was applied, nearly 70% of the wild-type plants were dead, whereas more than half of the AtARGAHs-knockout lines survived with relatively lower electrolyte leakage (Fig. 5A–C). On the contrary, more than 80% of the AtARGAHs-overexpressing lines were also dead, with higher electrolyte leakage than the wild type (Fig. 5A–C).

Additionally, at harvest, all mutant lines of AtARGAHs had significantly greater plant height and dry weight than the wild type under both water deficit and salt stress, whereas the overexpressing lines had much lower plant height and less dry weight compared with the wild type (Supplementary Fig. S2).

In summary, the expression repression of AtARGAHs displayed improved water deficit and salt tolerance, as confirmed by changes in physiological parameters including reduced electrolyte leakage, survival rate, water loss rate, and stomatal aperture. The relationship between the expression levels of AtARGAHs and plant tolerance to abiotic stress might be valuable for future crop breeding.

Freezing tolerance of AtARGAHs-overexpressing and -knockout lines

Besides water deficit and salt stresses, 2-week-old wild-type and different AtARGAHs plants were also used in a freezing tolerance test. After recovery from –6 °C for 7 d, all AtARGAHs mutant lines exhibited better leaf morphology and higher survival rate than the wild-type plants, whereas the AtARGAHs-overexpressing lines showed the lowest survival rate (Fig. 6A and B). As the lipid peroxidation product, MDA is an important indicator of oxidative damage (Shi et al., 2012b). Under freezing, MDA contents decreased in argah single and double mutants, but increased in the AtARGAHs-overexpressing lines when compared with the wild type. The changes in MDA content among the knockout mutants and overexpressing lines were correlated with the results of the freezing stress tolerance test (Fig. 6B and C).

Fig. 6.

Fig. 6.

Open in a new tab

Freezing tolerance of the wild type and the different AtARGAHs lines. (A) Phenotype of plants under control condition for 14 d and after 7 d recovery from freezing treatment. (B) Survival rate of the wild type and the different AtARGAHs lines under freezing stress condition. Survival rate was determined at 7 d after recovery from freezing stress test. (C) MDA content of the wild type and the different AtARGAHs lines under control conditions and at 7 d after 4 °C treatment. Values are mean ± SD of three independent experiments (this figure is available in colour at JXB online).

AtARGAHs modulate ROS metabolism under stress conditions

As two major indicators of ROS accumulation that can be induced by many environmental stresses, H2O2 and Inline graphic concentrations were determined through DAB and NBT staining, respectively. Under control conditions, no significant difference was observed among the wild type and the different AtARGAHs lines (Fig. 7A). When water deficit, salt, and freezing stresses were applied, less blue and brown spots for H2O2 and Inline graphic were stained in the seedlings of AtARGAHs mutant lines than the wild type, while deeper staining for H2O2 and Inline graphic was observed in the AtARGAHs-overexpressing lines (Fig. 7A). Consistently, the H2O2 quantification assay confirmed the results of DAB staining that argah mutant lines accumulated less H2O2 while the AtARGAHs-overexpressing lines exhibited higher H2O2 content when compared with the wild type under stress conditions (Fig. 7B).

Fig. 7.

Fig. 7.

Open in a new tab

ROS accumulation and antioxidant enzyme activities of the wild type and the different AtARGAHs lines under control and abiotic stress conditions. (A) DAB and NBT staining for H2O2 and Inline graphic concentrations in the leaves of plants under control and stress conditions. (B) Quantification of H2O2 content regulated by AtARGAHs lines under control and abiotic stress conditions. Fourteen-day-old plants were subjected to control conditions and water deficit and 150mM NaCl for 10 d, and 20-day-old plants were treated at 4 °C for 3 d. (C–E) Relative activities of SOD (C), CAT (D), and POD (E) quantified as fold-change relative to the wild type under control conditions. Values are mean ± SD of four independent experiments (this figure is available in colour at JXB online).

To analyse the correlation between the ROS accumulation and the activities of antioxidant enzymes in different AtARGAHs lines, the SOD, CAT, and POD activities were assayed under the same stress conditions. Under control conditions, no significant differences were observed among the different AtARGAHs lines and the wild-type plants (Fig. 7C–E). When subjected to water deficit, salt, and cold stresses, argah lines showed relatively lower SOD, CAT, and POD activities when compared with the wild-type plants, while the AtARGAHs-overexpressing lines exhibited significantly higher SOD, CAT, and POD activities relative to the wild type (Fig. 7C–E). These results indicated that the involvement of AtARGAHs in ROS homeostasis through regulation of antioxidant enzymes might be indirectly responsible for abiotic stress tolerance.

Discussion

Arginine is an essential metabolite in nitrogen distribution through its catabolism, which has clearly been shown that arginase, arginine decarboxylase, and NO synthase can metabolize arginine to other amino acids such as putrescine, spermidine, spermine, and proline (Flores et al., 2008; Brauc et al., 2012). Although the NO synthase protein has not been identified in plants except in two strains of the green alga Ostreococcus tauri, NO synthase-like activity has been detected in many higher plants, and inhibitors of mammalian NO synthase have been shown to inhibit NO generation in plants (Gupta et al., 2011; Shi et al., 2012b). Arginine supply could induce intracellular NO content in Arabidopsis, and Flores et al. (2008) concluded a model that posits a possible NO synthase-like activity, generating NO directly from arginine. In another recent report (Brauc et al., 2012), reduced arginase activity in the Arabidopsis transgenic lines also led to a significant increase in putrescine concentration for up to 40%. Previous research showed that polyamines could induce rapid biosynthesis of NO in root tip and primary leaves of Arabidopsis seedlings (Tun et al., 2006; Siddiqui et al., 2011; Wimalasekera et al., 2011). Therefore, the mutational elimination of AtARGAHs can conceivably make more arginine available for the synthesis of polyamines and NO, resulting in more polyamines and NO accumulation. This study confirmed the hypothesis that argah lines accumulated significantly higher concentrations of polyamines (putrescine and spermine) and NO, while AtARGAH-overexpressing lines exhibited lower arginine, putrescine, spermine, and NO concentrations relative to the wild type (Figs. 2–4). The large changes in polyamines and NO concentrations in AtARGAHs transgenic lines and mutants might result in changes at the physiological, biochemical, molecular, and cellular levels.

Interestingly, the AtARGAHs-knockout and -overexpressing lines showed relatively less arginine accumulation than the wild type (Fig. 2C). Consistently, Flores et al. (2008) found that argah1 and argah2 mutants had lower relative arginine pools. Brauc et al. (2012) also reported that both AtARGAH2-overexpressing and -silencing lines displayed significantly reduced relative arginine relative to the wild type. The decreased relative arginine in all AtARGAHs-overexpressing lines indicated a direct impact of AtARGAHs on arginine pools. More surprising was the decreased relative arginine in the AtARGAHs-knockout lines; Flores et al. (2008) speculated that localized arginine concentrations might be affected after mutation of AtARGAHs and proposed that more intramitochondrial or other specific localized arginine concentrations might be available for atargah mutants, which were further metabolized to NO and polyamines.

Both polyamines and NO function as stress signals in plant, mediating plant development and a range of biotic and abiotic stresses (Zhao et al., 2007; Neill et al., 2008; Lozano-Juste and Leόn, 2010; Wang et al., 2011b). NO may be a link between polyamine-mediated stress responses and other stress mediators, and multiple abiotic stresses may use NO as a mediator where polyamines are also involved (Tun et al., 2006; Neill et al., 2008). Recently, the present study group found that increasing NO content in Arabidopsis by expressing rat neuronal NO synthase conferred enhanced multiple stress tolerances (Shi et al., 2012a,b). These results together with others (Zhao et al., 2007, 2009; Corpas et al., 2009) indicated that proper levels of NO might be necessary and beneficial for plants in responses to several abiotic stresses and resistance against pathogenic bacteria. To confirm this hypothesis, the AtARGAHs-overexpressing and -knockout lines were used for abiotic stress tolerance test in this study. The underlying mechanisms were also dissected.

As shown in Figs. 5 and 6, the results indicated that AtARGAHs could modulate multiple abiotic stress tolerances. The expression repression of AtARGAHs in Arabidopsis exhibited improved water deficit, salt, and freezing tolerances, while overexpression of AtARGAHs showed the opposite effect on stress tolerances. All these effects were quite evident in the changes of physiological parameters including cell-membrane damage (electrolyte leakage), survival rate, water loss rate, stomatal aperture, and lipid peroxidation (MDA) (Figs. 5 and 6). Consistently, pharmacological studies and genetic results have both suggested that properly increased NO content resulted in enhanced biotic and abiotic stress tolerances, whereas NO-deficiency mutants were more sensitive to salt, freezing, and disease tolerance (Zhao et al., 2007, 2009; Shi et al., 2012a,b). Collectively, these observations emphasize the essential role of NO in plant stress response. Plants with proper NO levels could improve the tolerance to abiotic stress, while less NO levels may result in decreased stress tolerances. To date, many previously discovered mutants with altered NO levels, such as atnoa1, atnia1atnia2, and atnox1, have negative effects on plant development and yield even under control conditions, which may be attributed to unspecified roles in NO synthesis (He et al., 2004; Shi et al., 2012b). Interestingly, in the present study, AtARGAHs lines did not show any apparent negative effects on plant development (plant height and dry weight) under control conditions (Supplementary Fig. S2). Therefore, this report together with previous research (Shi et al., 2012a,b) may expand the possibilities for crop breeding through manipulating ARGAHs expression or in vivo NO levels.

Most of the environmental stresses such as water deficit, salt, and freezing can cause rapid and excessive accumulation of ROS, including H2O2, Inline graphic, and hydroxyl radical (·OH) (Zhu, 2002; Apel and Hirt, 2004; Mittler et al., 2004). ROS homeostasis is very important for plant stress tolerance because over production of ROS can lead to oxidative damage and cell death. In plant, abiotic stress-induced damage can be attenuated by different ways of inhibiting ROS accumulation (Mittler, 2002; Apel and Hirt, 2004; Mittler et al., 2004). Compared with the wild type, the weaker DAB and NBT staining for H2O2 and Inline graphic under stress conditions were observed in argah1, argah2, and argah1argah2 mutants (Fig. 7A and B), indicating less ROS accumulation of these mutants when subjected to abiotic stress conditions. Consistently, this study also observed that manipulation of AtARGAHs expression regulated the antioxidant enzyme activities including SOD, CAT, and POD under multiple abiotic stresses (Fig. 7C–E). Among these antioxidant enzymes, SOD catalyses Inline graphic into H2O2 and O2 at the first stage of the antioxidant defence system, while CAT and POD are also essential for break down of H2O2 (Mittler, 2002; Apel and Hirt, 2004; Mittler et al., 2004). All these antioxidant enzymes can protect plant cells from ROS injury and further help them to cope with different stresses. Thus, AtARGAHs may have important functions in modulating ROS accumulation and antioxidant enzyme activities, which are important components to alleviate oxidative stress-induced damages in plant responses to many environmental stresses.

As an important enzyme in arginine metabolism, arginine decarboxylase has been widely shown to be involved in abiotic stress responses (Wang et al., 2011a,b; Wimalasekera et al., 2011). In many plant species, including rice, tobacco, Arabidopsis, and tomato, overexpression of arginine decarboxylase that increases polyamine concentrations enhanced plant tolerance to water deficit, salt, and freezing stresses, partly due to its role in the regulation of ROS accumulation (Wang et al., 2011a,b; Wimalasekera et al., 2011). However, the role of arginase, another important enzyme involved in arginine metabolism, in abiotic stress remains largely unknown. In this study, the knockout mutants and AtARGAHs-overexpressing transgenic lines showed opposite effects on the accumulation of direct downstream arginine catabolism products including polyamines (putrescine and spermine) and NO (Figs. 3C and 4). These lines also showed opposite responses to water deficit, salt, and freezing stresses (Figs. 5 and 6), as well as opposite effect to maintain homeostasis of ROS in response to these stresses. Polyamines and NO may also have complex mechanisms to deal with ROS and other cellular responses derived from environmental stresses (Bright et al., 2006; Siddiqui et al., 2011; Wimalasekera et al., 2011). Application of NO donors can alleviate oxidative stresses induced by NaCl, chilling, iron deficiency, and seed desiccation by enhancing the activities of antioxidative enzymes and accumulation of osmolytes (Bethke et al., 2006; Liu et al., 2006; Sun et al., 2007; Bai et al., 2011; Khan et al., 2012).

Based on these observations, this study describes a model to depict the mechanism of AtARGAHs involved in abiotic stress responses (Fig. 8). As depicted, manipulating the expression of AtARGAHs modulates the specific localized arginine substrate for arginine decarboxylase or NO synthase and hence regulates endogenous polyamines and NO concentrations. The increased polyamines and NO modulate ROS accumulation through antioxidant enzyme activities and other adaptive responses. These in turn result in enhanced abiotic stress tolerance. Therefore, a new function of AtARGAHs in abiotic stress tolerance is indicated, which is, at least partly, attributed to its role in modulating polyamines, NO, and ROS concentrations. Additionally, both ARGAH and arginine decarboxylase are induced at the transcriptional level by abiotic stress including water deficit, salt, and freezing, supporting a role for arginine metabolism in abiotic stress responses. Interestingly, NO level is also affected by almost all abiotic stress conditions (Corpas et al., 2009; Zhao et al., 2009; Siddiqui et al., 2011; Shi et al., 2012b), indicating some links between arginine metabolism and NO level in response to abiotic stress.

Fig. 8.

Fig. 8.

Open in a new tab

Model depicting the mechanism of AtARGAHs involved in abiotic stress responses. Arginine can be metabolized by arginase, arginine decarboxylase, and NO synthase. Manipulating the expression of AtARGAHs modulates the arginine substrate for arginine decarboxylase or NO synthase, which in turn regulates endogenous polyamines and NO concentration. The increased polyamines and NO further modulate ROS accumulation through antioxidant enzyme activities and other adaptive responses, resulting in enhanced abiotic stress tolerance.

Taken together, this is the first report, as far as is known, indicating possible roles of AtARGAHs in abiotic stress. As water deficit, salt, and freezing are worldwide problems that seriously affect crop yields, genetic engineering of crops with enhanced multiple environmental adaptations may be possible by manipulating arginase expression.

Supplementary material

Supplementary data are available at JXB online.

Supplementary Fig. S1. Quantification of arginine and total free amino acids in 2-week-old plant leaves.

Supplementary Fig. S2. Effect of AtARGAHs expression on plant growth after water deficit and salt treatments.

Supplementary Table S1. Primers used for plasmid construction, semi-quantitative RT-PCR, and quantitative real-time PCR.

Supplementary Data
supp_64_5_1367__index.html (971B, html)

Acknowledgements

This research was supported by the Hundred Talents Program, the Knowledge Innovative Key Program of Chinese Academy of Sciences (grant no. 54Y154761O01076 to Zhulong Chan), and the National Natural Science Foundation of China (grant no. 31200194 to Haitao Shi). The authors thank Prof Yingtang Lu, Dr Rongjun Li, Ms Wen Liu, and Ms Wei Cai for their help in this research.

References

  1. Apel K, Hirt H. 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology 55, 373–399 [DOI] [PubMed] [Google Scholar]
  2. Bai X, Yang L, Tian M, Chen J, Shi J, Yang Y, Hu X. 2011. Nitric oxide enhances desiccation tolerance of recalcitrant Antiaris toxicaria seeds via protein S-nitrosylation and carbonylation. PLoS ONE 6, e20714 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bethke PC, Libourel IG, Jones RL. 2006. Nitric oxide reduces seed dormancy in Arabidopsis . Journal of Experimental Botany 57, 517–526 [DOI] [PubMed] [Google Scholar]
  4. Bradford NM. 1976. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254 [DOI] [PubMed] [Google Scholar]
  5. Brauc S, Vooght ED, Claeys, Geuns JMC, Höfte M, Angenon G. 2012. Overexpression of arginase in Arabidopsis thaliana influences defence responses against Botrytis cinerea . Plant Biology 14, 39–45 [DOI] [PubMed] [Google Scholar]
  6. Bright J, Desikan R, Hancock JT, Weir IS, Neill SJ. 2006. ABA induced no generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. The Plant Journal 45, 113–122 [DOI] [PubMed] [Google Scholar]
  7. Brownfield DL, Todd CD, Deyholos MK. 2008. Analysis of Arabidopsis arginase gene transcription patterns indicates specific biological functions for recently diverged paralogs. Plant Molecular Biology 67, 429–440 [DOI] [PubMed] [Google Scholar]
  8. Chan ZL, Grumet R, Loescher W. 2011. Global gene expression analysis of transgenic, mannitol producing, and salt-tolerant Arabidopsis thaliana indicates widespread changes in abiotic and biotic stress-related genes. Journal of Experimental Botany 62, 4787–4803 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen H, McCaig BC, Melotto M, He SY, Howe JA. 2004. Regulation of plant arginase by wounding, jasmonate, and the phytotoxin coronatime. Journal of Biological Chemistry 279, 45998–46007 [DOI] [PubMed] [Google Scholar]
  10. Corpas FJ, Hayashi M, Mano S, Nishimura M, Barroso JB. 2009. Peroxisomes are required for in vivo nitric oxide accumulation in the cytosol following salinity stress of Arabidopsis plants. Plant Physiology 151, 2083–2094 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR. 2000. Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proceedings of the National Academy of Sciences, USA 97, 3718–3723 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fisher GH, Arias I, Quesada I, D’Aniello S, Errico F, Di Fiore MM, D’Aniello A. 2001. A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues. Amino Acids 20, 163–173 [DOI] [PubMed] [Google Scholar]
  13. Flores T, Todd CD, Tovar-Mendez A, Dhanoa PK, Correa-Aragunde N, Hoyos ME, Brownfield DM, Mullen RT, Lamattina L, Polacco JC. 2008. Arginase-negative mutants of Arabidopsis exhibit increased nitric oxide signaling in root development. Plant Physiology 147, 1936–1946 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gravot A, Deleu C, Wagner G, Lariagon C, Lugan R, Todd C, Wendehenne D, Delourme R, Bouchereau A, Manzanares-Dauleux MJ. 2012. Arginase induction represses gall development during clubroot infection in Arabidopsis . Plant and Cell Physiology 53, 901–911 [DOI] [PubMed] [Google Scholar]
  15. Gupta KJ, Fernie AR, Kaiser WM, Dongen JT. 2011. On the origins of nitric oxide. Trends in Plant Science 16, 160–168 [DOI] [PubMed] [Google Scholar]
  16. He Y, Tang RH, Hao Y, et al. 2004. Nitric oxide represses the Arabidopsis floral transition. Science 305, 1968–1971 [DOI] [PubMed] [Google Scholar]
  17. Khan MN, Siddiqui MH, Firoz Mohammad, Naeem M. 2012. Interactive role of nitric oxide and calcium chloride in enhancing tolerance to salt stress. Nitric Oxide 27, 210–218 [DOI] [PubMed] [Google Scholar]
  18. Krumpelman PM, Freyermuth SK, Cannon JC, Fink GR, Polacco JC. 1995. Nucleotide sequence of Arabidopsis thaliana arginase expressed in yeast. Plant Physiology 107, 1479–1480 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liu Y, Jiang H, Zhao Z, An L. 2006. Nitric oxide synthase like activity-dependent nitric oxide production protects against chilling-induced oxidative damage in Chorispora bungeana suspension cultured cells. Plant Physiology and Biochemistry 48, 936–944 [DOI] [PubMed] [Google Scholar]
  20. Lozano-Juste J, Leόn J. 2010. Enhanced abscisic acid-mediated responses in nia1nia2noa1-2 triple mutant impaired in NIA/NR- and AtNOA1-dependent nitric oxide biosynthesis in Arabidopsis . Plant Physiology 152, 891–903 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ma X, Cheng Z, Qin R, et al. 2012. OsARG encodes an arginase that plays critical roles in panicle development and grain production in rice. The Plant Journaldoi: 10.1111/j.1365-313X.2012.05122.x (E-pub ahead of print). [DOI] [PubMed] [Google Scholar]
  22. Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7, 405–410 [DOI] [PubMed] [Google Scholar]
  23. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F. 2004. Reactive oxygen gene network of plants. Trends in Plant Science 9, 490–498 [DOI] [PubMed] [Google Scholar]
  24. Murphy ME, Noack E. 1994. Nitric oxide assay using hemoglobin method. Methods in Enzymology 233, 240–250 [DOI] [PubMed] [Google Scholar]
  25. Neill S, Bright J, Desikan R, Hancock J, Harrison J, Wilson I. 2008. Nitric oxide evolution and perception. Journal of Experimental Botany 59, 25–35 [DOI] [PubMed] [Google Scholar]
  26. Sastry CSP, Tummuru MK. 1984. Spectrophotometric determination of arginine in proteins. Food Chemistry 15, 257–260 [Google Scholar]
  27. Shi HT, Li RJ, Cai W, Liu W, Fu ZW, Lu YT. 2012a. In vivo role of nitric oxide in plant response to abiotic and biotic stress. Plant Signaling and Behavior 7, 438–440 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shi HT, Li RJ, Cai W, Liu W, Wang CL, Lu YT. 2012b. Increasing nitric oxide content in Arabidopsis thaliana by expressing rat neuronal nitric oxide synthase resulted in enhanced stress tolerance. Plant and Cell Physiology 53, 344–357 [DOI] [PubMed] [Google Scholar]
  29. Shi HT, Wang YP, Cheng ZM, Ye TT, Chan ZL. 2012c. Analysis of natural variation in bermudagrass (Cynodon dactylon) reveals physiological responses underlying drought tolerance. PLoS ONE 7, e53422 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Siddiqui MH, Al-Whaibi MH, Basalah MO. 2011. Role of nitric oxide in tolerance of plants to abiotic stress. Protoplasma 248, 447–455 [DOI] [PubMed] [Google Scholar]
  31. Sun B, Jing Y, Chen K, Song L, Chen F, Zhang L. 2007. Protective effect of nitric oxide on iron deficiency-induced oxidative stress in maize (Zea mays). Journal of Plant Physiology 164, 536–543 [DOI] [PubMed] [Google Scholar]
  32. Tun NN, Santa-Catarina C, Begum T, Silveira V, Handro W, Floh EI, Scherer GF. 2006. Polyamines induce rapid biosynthesis of nitric oxide (NO) in Arabidopsis thaliana seedlings. Plant and Cell Physiology 47, 346–54 [DOI] [PubMed] [Google Scholar]
  33. Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu JK. 2006. Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. The Plant Journal 45, 523–539 [DOI] [PubMed] [Google Scholar]
  34. Walter HJP, Geuns JMC. 1987. High speed HPLC analysis of polyamines in plant tissues. Plant Physiology 83, 232–234 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wang J, Sun PP, Chen CL, Wang Y, Fu XZ, Liu JH. 2011a. An arginine decarboxylase gene PtADC from Poncirus trifoliate confers abiotic stress tolerance and promotes primary root growth in Arabidopsis . Journal of Experimental Botany 62, 2899–2914 [DOI] [PubMed] [Google Scholar]
  36. Wang BQ, Zhang QF, Liu JH, Li GH. 2011b. Overexpression of PtADC confers enhanced dehydration and drought tolerance in transgenic tobacco and tomato: effect on ROS elimination. Biochemical and Biophysical Research Communications 413, 10–16 [DOI] [PubMed] [Google Scholar]
  37. Wimalasekera R, Tebartz F, Scherer GF. 2011. Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses. Plant Science 18, 593–603 [DOI] [PubMed] [Google Scholar]
  38. Zhao MG, Chen L, Zhang LL, Zhang WH. 2009. Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis . Plant Physiology 151, 755–767 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhao MG, Tian QY, Zhang WH. 2007. Nitric oxide synthase-dependent nitric oxide production is associated with salt tolerance in Arabidopsis . Plant Physiology 144, 206–217 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhu JK. 2002. Salt and drought stress signal transduction in plants. Annual Review of Plant Biology 53, 247–273 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
supp_64_5_1367__index.html (971B, html)
supp_ers400_Supplementary_data_of_JEXBOT_2012___092528__revised_.pdf (321.8KB, pdf)

Articles from Journal of Experimental Botany are provided here courtesy of Oxford University Press

RESOURCES