SEARCH

SEARCH BY CITATION

Keywords:

  • Arabidopsis thaliana;
  • chilling;
  • nitrate reductase;
  • nitric oxide;
  • phosphatidic acid;
  • sphingolipids

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Chilling triggers rapid molecular responses that permit the maintenance of plant cell homeostasis and plant adaptation. Recent data showed that nitric oxide (NO) is involved in plant acclimation and tolerance to cold. The participation of NO in the early transduction of the cold signal in Arabidopsis thaliana was investigated.
  • The production of NO after a short exposure to cold was assessed using the NO-sensitive fluorescent probe 4, 5-diamino fluoresceine diacetate and chemiluminescence. Pharmacological and genetic approaches were used to analyze NO sources and NO-mediated changes in cold-regulated gene expression, phosphatidic acid (PtdOH) synthesis and sphingolipid phosphorylation.
  • NO production was detected after 1–4 h of chilling. It was impaired in the nia1nia2 nitrate reductase mutant. Moreover, NO accumulation was not observed in H7 plants overexpressing the A. thaliana nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1). Cold-regulated gene expression was affected in nia1nia2 and H7 plants. The synthesis of PtdOH upon chilling was not modified by NO depletion. By contrast, the formation of phytosphingosine phosphate and ceramide phosphate, two phosphorylated sphingolipids that are transiently synthesized upon chilling, was negatively regulated by NO.
  • Taken together, these data suggest a new function for NO as an intermediate in gene regulation and lipid-based signaling during cold transduction.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Cold stress is one of the most common adverse situations experienced by plants during their lifespan and restricts the spread of nontolerant plants in temperate climates. The ability of plants to cope with chilling conditions is dependent upon the presence of appropriate adaptative responses that prevent the deleterious effects of chilling on membranes and cellular metabolism. Even more importantly, the exposure of tolerant plants to nonfreezing temperatures allows them to cold-acclimate and subsequently tolerate subzero temperatures. The response to chilling involves a remodeling of cellular metabolism, including the accumulation of cryoprotective compounds such as proline and sugars (Cook et al., 2004; Kaplan et al., 2004). This remodeling at least partially results from the expression of specific genes (Cook et al., 2004). Large-scale transcriptomic analyses have identified several hundreds of genes the expression of which is regulated by cold (Provart et al., 2003; Lee et al., 2005; Vergnolle et al., 2005; Vogel et al., 2005). A large set of these genes are controlled by C-repeat binding factors (CBFs) which bind to the common C-repeat motif CCGAC found in their promoters (reviewed in Ruelland et al., 2009). The expression of CBF genes is itself responsive to cold and occurs rapidly (15 min) following chilling (Liu et al., 1998). Moreover, the CBF pathway is finely tuned through a set of additional transcription factors either positively Inducer of CBF Expression1 (ICE1) or negatively (MYB15, ZAT12, etc.) regulating CBF expression (Ruelland et al., 2009). Adding to the complexity of the regulatory network operating during the cold response, only 10–15% of cold-responsive genes belong to the CBF cluster, which indicates the existence of yet undiscovered regulators (Hannah et al., 2005).

How the cold signal is transduced from membranes towards the nucleus, where it triggers gene expression, has been the focus of numerous studies and led to the characterization of cellular signals operating within the cold transduction cascade (for a review, see Ruelland et al., 2009). In this context, lipids or lipid-derived molecules are important participants in the cold response, as observed in other biotic and abiotic stress conditions (Meijer & Munnik, 2003). For instance, variations in the quantity of the phospholipid-derived signal phosphatidic acid (PtdOH) occur within the first 1 min of cold exposure in Arabidopsis thaliana culture cells, which indicates that PtdOH production is one of the earliest response of plants to cold (Ruelland et al., 2002). PtdOH can be produced by the direct hydrolysis of membrane phospholipids by phospholipase D (PLD). It can also originate from the action of phospholipase C (PLC) which generates inositol triphosphate and diacyglycerol (DAG), and the subsequent phosphorylation of DAG by DAG kinases (DGKs). Ruelland et al. (2002) showed that both pathways participate in PtdOH production in chilled plants. Interestingly, the PLC/DGK- and PLD-derived PtdOH pools appear to participate in distinct signaling pathways leading to the activation of distinct cold-responsive gene clusters (Vergnolle et al., 2005). Although modifications of the pools of precursors or intermediates of PtdOH synthesis such as phosphatidylinositol phosphate (PtdInsP), PtdInsP2 and IP3 have been reported, their function during the cold response remains unknown (Ruelland et al., 2002). In addition to phospholipid-derived molecules, sphingolipids were recently demonstrated to be potent signals in plants (Lynch et al., 2009; Pata et al., 2010). In particular, the intermediates of complex membrane sphingolipid synthesis, that is, long-chain bases (LCB) and ceramides (Cer) together with their phosphorylated counterparts, participate in the plant response to abscisic acid or pathogens (Ng et al., 2001; Coursol et al., 2003, 2005; Liang et al., 2003). Several studies have shown differences in the membrane structural sphingolipid abundance/composition of acclimated vs nonacclimated plants and cold-tolerant vs cold-sensitive species (Lynch & Steponkus, 1987; Kawaguchi et al., 2000). We recently observed that chilling also resulted in the rapid and transient formation of two particular phosphosphingolipids (C. Dutilleul & I. Guillas, unpublished data). The less hydrophobic species was identified as phytosphingosine phosphate (PHS-P) as it co-migrated with commercial PHS-P when analyzed by TLC and high-performance liquid chromatography (HPLC) following derivatization of the amine group with the fluorescent reagent o-phthaldialdehyde The most hydrophobic species was identified as a ceramide phosphate as it could be derivatized with o-phthaldialdehyde only after freeing the amino group of the LCB from the acyl moiety by strong acid hydrolysis. Subsequently, the derivatized LCB co-migrated with 4-hydroxy-8- sphingenine (PHSe), indicating that the overall molecule corresponds to a PHSe-based ceramide phosphate. These data therefore suggest that specific phosphosphingolipids such as PHS-P and ceramide phosphate (Cer-P) may participate in cold transduction.

Previous data have indicated the involvement of nitric oxide (NO) in plant acclimation and freezing tolerance (Zhao et al., 2009). NO is a small gaseous signaling molecule participating in plant development and in responses to abiotic and biotic stresses (Besson-Bard et al., 2008; Neill et al., 2008). Two major pathways have been implicated in NO production in plants (reviewed in Besson-Bard et al., 2008). On the one hand, NO can be produced during the conversion of arginine to citrulline by NO synthase-like activities, although the existence of such enzymes in plants is still a matter of controversy. On the other hand, nitrate reductases (NRs) are potent sources of NO, as indicated by the absence of NO synthesis in the A. thaliana nia1nia2 NR double mutant during several stress responses. Upon the exposure of A. thaliana to 4°C, NO production mainly dependent on NR activity was observed after 1 to 14 d of stress (Zhao et al., 2009). The nia1nia2 NR double mutant defective in NO production is also impaired in freezing tolerance and cold acclimation (Zhao et al., 2009). Interestingly, Zhao et al. (2009) showed that cold-induced NO participates in the regulation of proline synthesis by regulating genes of the proline biosynthetic pathway, which may be a function of NO in freezing tolerance. The synthesis of NO in response to low temperatures may be a general feature in plants as it has also been reported in Lotus japonicus and Pisum sativum (Shimoda et al., 2005; Corpas et al., 2008). In this last case, NO production originated from an NO synthase-like activity, which suggests that diverse NO sources may be involved, depending on plant species or chilling conditions. Despite the demonstration of a key role for NO in cold stress acclimation and freezing tolerance, little is known about its possible occurrence and function during the earlier steps of the plant response to chilling. A recent study reported that modifications of the pattern of nitrosylated proteins occurred within the first few hours of Brassica juncea exposure to 4°C, which indirectly suggests that NO production also occurs rapidly after chilling exposure (Abat & Deswal, 2009). Nevertheless, the identity of the target proteins whose nitrosylation was cold-modified did not provide significant insights into how NO may participate in cold transduction. In the light of the prominent function of lipid signaling in the early cold response, a function for NO in regulating the formation of lipid-derived signals is possible. Strengthening this hypothesis, Laxalt et al. (2007) showed that NO is a regulator of PtdOH synthesis in plants. On the one hand, exogenous treatments with NO donors led to PtdOH synthesis in tomato (Solanum lycopersicum), Vicia faba and cucumber (Cucumis sativus) (Laxalt et al., 2007; Distefano et al., 2008; Lanteri et al., 2008). On the other hand, PtdOH synthesis in response to xylanase and auxins was impaired by treatments with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (Laxalt et al., 2007; Lanteri et al., 2008). Interestingly, both the PLC/DGK and PLD pathways can be regulated by NO, although such regulation may depend on the enzyme isoforms responsible for PtdOH synthesis (Laxalt et al., 2007; Distefano et al., 2008).

In the present paper, we investigated the putative functions of NO in the transduction of the cold signal in A. thaliana. We provide evidence that NO is produced within the first few hours following cold exposure. NO production depended on the activity of NR and could be impaired by the over-expression of the nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1). Moreover, genetic impairment of NO accumulation upon chilling inhibited the expression of specific cold-responsive genes. We also found that NO was not involved in PLD- or PLC/DGK-dependent cold-induced PtdOH synthesis. However, we provide evidence that the formation of PHS-P and Cer-P which rapidly occurs upon cold exposure was regulated by endogenous NO concentrations. Taken together, our data support a role for NO in the early transduction of the cold signal.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material and treatment conditions

Experiments were performed using Arabidopsis thaliana L. Heynh. wild type (WT) in the Columbia (Col-0) background as cultured cells, seedlings and plants. The cell suspensions were cultivated and cold-treated as described by Ruelland et al. (2002).

Plants of the wild-type A. thaliana ecotype Col-0, the nia1nia2 NR mutant (Wilkinson & Crawford, 1993) and the nonsymbiotic hemoglobin AHb1-overexpressing line (H7) (Perazzolli et al., 2004) were grown for 4 wk in a growth chamber (8 h : 16 h dark : light photoperiod; 100 μmol m−2 s−1; 70% humidity) in a peat : perlite mixture (2 : 1). When testing the effect of cPTIO on cold-regulated gene expression, fully expanded leaves were infiltrated on whole plants with a syringe and kept at 22°C for 2 h for complete mesophyll drying before exposure to cold.

Alternatively, seeds were sterilized twice with diluted bleach and rinsed with 95% ethanol. When dry, seeds were sown on basic half-strength Murashige and Skoog (MS) medium, pH 5.7 (M0221; Sigma-Aldrich, Lyon, France), supplemented with 10 g l−1 sucrose and 8 g l−1 agar and stratified for 2 d at 4°C. Plates were subsequently placed in a growth chamber, under continuous light (100 μmol m−2 s−1) at 22°C. For the nia1nia2 line, the medium was supplemented with 10 mM ammonium succinate.

For cold treatment, plants were transferred to a cold room set to 4°C, under the same light conditions.

Chemicals

4,5-Diaminofluoresceine diacetate (DAF2-DA) and sodium nitroprusside (SNP) were purchased from Sigma-Aldrich. NG-Nitro-l-arginine-methyl ester (l-NAME), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), nitroso-gluthatione (GSNO), S-nitroso-N-acetyl-D,L-penicillamine (SNAP), 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA NONOate) and Griess reagent were purchased from Alexis Biochemicals (Lausen, Switzerland).

Fluorimetric measurement of NO

Experiments were carried out on 4-wk-old plants. Following low-temperature exposure, leaf disks (8 mm) were excised and vacuum-infiltrated for 3 min with 3 ml of infiltration buffer IB (10 mM Tris buffer (pH 7.4) and 10 mM KCl) containing 10 μM DAF2-DA. When indicated, IB was supplemented with different inhibitors during the infiltration step. Disks were subsequently washed in the dark for 30 min at 22°C with IB buffer. Individual disks were placed in a 96-well plate containing 200 μl of IB buffer per well. Fluorescence was immediately measured (λexc = 488 nm; λem = 515 nm) using a Carry Eclipse fluorometer (Varian, Palo Alto, CA, USA). Typical experiments had at least six infiltrated disks per condition tested.

Measurement of NO by chemiluminescence

NO measurement by chemiluminescence is based on the specific reaction of NO gas with ozone to form activated NO2 which emits luminescence during its transition to steady state. Following chilling, three plants (4 wk old; roots in water) were placed in a transparent lid chamber with 1 l air volume. A constant flow of measuring gas (purified air or nitrogen) of 1.5 l min−1 was pulled through the chamber and subsequently through the chemiluminescence detector (CLD 770 AL ppt; Eco-Physics, Dürnten, Switzerland; detection limit 20 ppt; 20 s time resolution) by a vacuum pump connected to an ozone destroyer. The ozone generator of the chemiluminescence detector was supplied with dry oxygen (99%). The measuring air was made NO-free by passing it through a custom-made charcoal column (1 m long; internal diameter 3 cm; particle size 2 mm). Calibration was routinely carried out with NO-free air (0 ppt NO) and with various concentrations of NO (1–35 ppb) adjusted by mixing the calibration gas (500 ppb NO in nitrogen; Messer Griesheim, Darmstadt, Germany) with NO-free air. Flow controllers (FC-260; Tylan General, Eching, Germany) were used to adjust all gas flows. Data were analyzed with a custom-made program using Visual Designer™ (Intelligent Instrumentation Inc., Tucson, AZ, USA).

Nitrite content

Leaves (c. 100 mg) were ground in 500 μl of deionized water. Following 30 min of centrifugation at 10 000 g, 250 μl of supernatant was mixed with an equal volume of Griess reagent (Alexis Biochemicals), and absorbance was measured at 530 nm. NaNO2 was used as a standard for NO2 quantification.

RT-PCR analysis

RNAs were extracted from 2-wk-old plantlets or infiltrated leaves with Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. One microgram of total RNA was treated with DNase I and subsequently reverse-transcribed using the Omniscript RT kit (Qiagen, Valencia, CA, USA). Five time-diluted RT products were used for PCR analysis, as described previously (Vergnolle et al., 2005). For AHb1 amplification, primers were: AHb1-F: 5′-GTA-GTG-AAG-TCT-TGG-AGT-GT-3′ and AHb1-R: 5′-CAC-CGT-ATT-TAG-AAT-GGC-TG-3′. Other primers used have been previously described (Vergnolle et al., 2005).

Analysis of PtdOH production

Labeling and cold treatment of cultured cells were performed as described in Ruelland et al. (2002), with labeling started 15 min (short term) or 16 h (long term) before the cold treatment. Pharmacological agents were added to cell cultures 15 min before the cold treatment. When PLD-dependent PtdOH was analyzed, 10 mM unlabeled phosphate was added 15 min before cold treatment. PtdOH was extracted as follows. First, 0.7 ml of 5% trichloracetic acid was added to the cell suspensions. Cells were then washed twice with cold water. Lipids were extracted by incubation at 65°C for 15 min, successively in methanol, choroform : methanol (1 : 1, v/v) and chloroform. Pooled extracts were dried under nitrogen and re-suspended in methanol. Total lipids were developed by thin-layer chromatography (TLC) on Silica 60 plates (Merck, Darmstadt, Germany) using solvent system 1 (SS1; chloroform : methanol : ammonia 25% (m/v) : water (90 : 70 : 4 : 16); Munnik et al., 1994). Radiolabeled PtdOH was revealed and quantified using a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA).

Analysis of phosphosphingolipids

Total lipids were extracted as for PtdOH analysis. They were then treated for 1 h at 65°C in NH4OH : methanol (1 : 1, v/v) (Reggiori & Conzelmann, 1998), and phase-extracted in acetic acid : water : chloroform (7 : 3 : 3). Nitrogen-dried sphingolipids were re-suspended in methanol. Incorporated radioactivity was measured by liquid scintillation. Sphingolipids were developed by thin-layer chromatography on Silica 60 plates (Merck) using solvent system 2 (SS2; chloroform : acetone : methanol : acetic acid : water (10 : 4 : 3 : 2 : 1); Coursol et al., 2005).

For plantlet labeling, 2-wk-old plantlets were transferred to flasks containing liquid half-strength MS medium. [33P]-orthophosphate (53 MBq l−1) was added to each flask 15 min before cold treatment. Sphingolipids were subsequently extracted, purified and analyzed as for cultured cells. Radiolabeled sphingolipids were revealed and quantified using a Storm PhosphorImager (Molecular Dynamics).

Statistical analysis

Results are presented as mean values ± standard deviations. Mean comparisons were calculated by Student’s test with P-values indicated in figure legends.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

NO accumulates at early stages of the A. thaliana cold response

To examine the possible production of NO in response to low temperature, NO was detected in control and chilled A. thaliana plants using the permeant NO-sensitive fluorescent probe DAF2-DA, which detects oxidized forms of NO. Four-wk-old plants, which have a sufficient number of fully expanded leaves and sufficiently large biomass for our purposes, were used for these experiments. As shown in Fig. 1(a), a twofold increase in fluorescence was observed after 1 h of cold exposure, whereas fluorescence was not modified in leaf disks from plants kept at 22°C. The fluorescence was slightly higher after 4 h of cold exposure (2.25-fold). The NO scavenger cPTIO efficiently impaired the fluorescence increase triggered by cold, strongly suggesting that the fluorescence observed corresponded to NO detection (Fig. 1a). In parallel, NO measurements were carried out on whole 4-wk-old plants using chemiluminescence detection. As shown in Fig. 1(b), an increase in the rate of NO production was observed in plants stressed for 4 h at 4°C, whereas it remained steady and low in unstressed plants. It continuously increased within the first hour of measurement to reach a plateau of 0.15 nmol h−1 g−1 FW compared with the 0.035 nmol h−1 g−1 FW measured in plants kept at 22°C (Fig. 1b). These observations confirm that NO measurements in leaf disks reflect bona fide NO production triggered by cold. Taken together, these data extend the occurrence NO production observed in previous studies to the early stages of the A. thaliana response to cold stress.

image

Figure 1.  Nitric oxide (NO) is produced in Arabidopsis thaliana leaves upon chilling. (a) Cold-induced DAF2-DA fluorescence in chilled and control leaf disks. Leaf disks were harvested from control or chilled 4-wk-old plants at the indicated times following cold exposure and loaded with 4,5-diaminofluoresceine diacetate (DAF2-DA). When indicated, 1 mM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) was loaded together with DAF2-DA. Values are the mean of individual disk fluorescence, expressed in arbitrary units (AU), and standard error (n = 16–24). Asterisks represent statistically significant differences between chilled plants and the corresponding control; *, < 0.05; ***, < 0.001. (b) NO emission rate of chilled (gray trace) and control (dark trace) plants following exposure at 4°C or 22°C for 4 h. Traces show the NO emission rate measured for 75 min in the dark and are representative of four replicates. Note that results represent rates of NO emission.

Download figure to PowerPoint

NO production requires NR activity

In plants, NO production has mainly been associated with arginine-dependent or NR-dependent pathways. To examine the relative contributions of these sources to the early generation of NO following chilling, we analyzed the effects of inhibitors of each pathway. As shown in Fig. 2(a), infiltration with either 1 mM tungstate, an NR inhibitor, or 1 mM l-NAME, an arginine analog that inhibits mammalian NO synthase activity, did not significantly modify fluorescence levels in leaf disks from unstressed plants. NO production in leaf disks from chilled plants was not affected by l-NAME while it was strongly inhibited (by 68%) by tungstate. As tungstate was previously reported to be a rapid and potent inhibitor of NR activity in vitro and in vivo (Bright et al., 2006), these data suggested that NR-dependent processes were involved in early NO production of chilled A. thaliana. As the possibility of unspecific effects of tungstate cannot be excluded, NO production was compared in unstressed and cold-stressed WT and nia1nia2 NR double mutant plants. As shown in Fig. 2(b), unstressed nia1nia2 plants showed lower NO production than WT plants. Upon chilling, no increase in NO production was observed in nia1nia2 plants. Moreover, cold stress exposure led to a modification of plant nitrite content. As shown in Fig. 2(c), the nitrite concentration was significantly increased by low-temperature treatment depending on the duration of stress. This increase was not observed in leaves pre-infiltrated with 1 mM tungstate (data not shown). Taken together, these results are consistent with the NR-dependent pathway being a source of NO in response to chilling.

image

Figure 2.  Nitrate reductase (NR) participates in cold-induced nitric oxide (NO) production in Arabidopsis thaliana. (a) Effect of NR and NO synthase inhibitors on cold-induced NO production. Following plant treatment for 4 h at 22°C (open bars) or 4°C (closed bars), leaf disks were infiltrated with 4,5-diaminofluoresceine diacetate (DAF2-DA) in the absence or presence of 1 mM tungstate or 1 mM NG-nitro-l-arginine-methyl ester (l-NAME). Values are the mean of individual disk fluorescence, expressed in arbitrary units (AU), and standard error (n = 20–22). Asterisks represent statistically significant differences between the control and the corresponding inhibitor-treated disks; ***, < 0.001. (b) NO production in the NR double mutant nia1nia2. Following plant treatment for 4 h at 22°C (open bars) or 4°C (closed bars), leaf disks were infiltrated with DAF2-DA. Values are the mean of individual disk fluorescence, expressed in arbitrary units, and standard error (n = 18). Asterisks represent statistically significant differences between nia1nia2 and the corresponding wild-type (WT) disks; *, < 0.05; ***, < 0.001. (c) Nitrite content of stressed and unstressed leaves. Nitrite content was measured in leaves from unstressed (22°C) or stressed (4°C) plants after different cold exposure durations. Values are the mean and standard error (n = 5). Asterisks represent statistically significant differences between control and chilled plants; *, < 0.05.

Download figure to PowerPoint

Overexpression of the nonsymbiotic hemoglobin AHb1 impairs cold stress-induced NO production

Recent data have highlighted the function of the plant nonsymbiotic hemoglobin AHb1 in regulating NO bioactivity by oxidizing bioactive NO into inactive NO3 (Perazzolli et al., 2004). To determine whether AHb1 retains such a function during the cold stress response, we analyzed the production of NO in an A. thaliana line over-expressing AHb1 (H7). As shown in Fig. 3(a), leaf disks from H7 plants showed a lower level of DAF fluorescence at 22°C. When plants were exposed to low temperatures, NO production was strongly impaired in H7 plants compared with the WT, indicating that AHb1 dissipated the NO signal generated upon cold exposure. Interestingly, although it was lower than that of WT plants, the difference in fluorescence level between unstressed and chilled H7 plants remained significant, which indicates that they retain their capacity to produce NO. We further examined the expression of AHb1 in WT Col-0 plants following cold exposure. As shown in Fig. 3(b), a low level of transcripts was detected in unstressed plants. A significant accumulation was observed after 24 h at 4°C. This accumulation was reduced when leaves were pre-infiltrated with cPTIO (Fig. 3b), suggesting that AHb1 expression could be triggered by the early production of NO and that AHb1 could subsequently participate in the modulation of cold-induced NO concentration in planta.

image

Figure 3.  Over-expression of the nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1) lowers nitric oxide (NO) concentrations in cold-stressed Arabidopsis thaliana plants. (a) The NO content of the AHb1-over-expressing line H7. Following plant treatment for 4 h at 22°C (open bars) or 4°C (closed bars), leaf disks were infiltrated with 4,5-diaminofluoresceine diacetate (DAF2-DA). Values are the mean of individual disk fluorescence and standard error (n = 12). Asterisks represent statistically significant differences between the wild-type (WT) and the corresponding H7 disks; ***, < 0.001. (b) Expression of the AHb1 gene in chilled WT leaves. AHb1 transcripts were detected by RT-PCR in attached leaves of 4-wk-old Columbia (Col-0) plants infiltrated with H2O or 1 mM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and cold-treated for the indicated times. S19 transcripts were used as an internal standard.

Download figure to PowerPoint

NO participates in CBF-dependent cold-induced gene expression

To gain insights into possible targets for NO during the cold response, we analyzed the expression of cold-responsive genes in chilled nia1nia2 and H7 plants. We therefore selected a subset of gene markers including transcription factors such as CBFs and ZAT12, and cold-regulated genes Cold Regulated 15a gene (COR15a), Low temperature induced gene 30 (LT130) and LTI78). As shown in Fig. 4, the transcripts of the different selected genes rapidly accumulated in response to low-temperature exposure in WT plants. The accumulation of CBF1 and CBF3 transcripts was impaired in both nia1nia2 and H7 lines. Interestingly, the expression of CBF2 was not affected by NO depletion and may therefore indicate different regulation of CBF members by NO. Similar regulation was observed in 4-wk-old plant leaves infiltrated with 1 mM cPTIO before cold exposure (Supporting Information, Fig. S1). The lower CBF1 and CBF3 expression in H7 and nia1nia2 lines was correlated with impairment of the expression of the COR15a, LTI30 and LTI78 genes, which have been described as CBF targets. We observed that, although the different genes tested were still responsive to cold, supplementation of the growth medium with ammonium modified the kinetics and/or magnitude of their expression in Col-0 plantlets (Fig. 4). However, the expression of ZAT12, a transcription factor that does not belong to the CBF family, was not affected by NO depletion. Taken together, these results indicate that NO participates in gene regulation through a CBF-dependent pathway during the low-temperature response.

image

Figure 4.  Expression of cold-regulated genes in chilled nia1nia2 and H7 plants. Two-wk-old Arabidopsis thaliana plantlets were transferred at 4°C and harvested at the indicated time. C-repeat binding factor 1 (CBF1), CBF2, CBF3, Cold Regulated 15a gene (COR15a), Low temperature induced gene 30 (LTI30), Low temperature induced gene 78 (LTI78) and ZAT12 transcripts were detected by RT-PCR using specific primers. A specific control (Col-0/NH4+) corresponding to nia1nia2 growth conditions (medium supplemented with 10 mM ammonium succinate) was used for comparison with the nia1nia2 line. S19 transcripts were used as an internal standard. Expression patterns are representative of three independent biological experiments.

Download figure to PowerPoint

NO is not implicated in the regulation of cold-induced phosphatidic acid production

As NO is rapidly produced in response to cold stress and participates in gene expression, we investigated its contribution to early cold signal transduction. In this context, we first addressed the possible involvement of NO in regulating cold-induced PtdOH production. NO was recently reported to regulate the PLC and PLD pathways which are involved in PtdOH synthesis in plants in a range of responses to stress (Laxalt et al., 2007; Distefano et al., 2008; Lanteri et al., 2008). Therefore, PtdOH originating from either the PLC or PLD pathway was monitored by in vivo labeling of PtdOH in A. thaliana cell cultures incubated with [33P]-orthophosphate before cold shock, as previously described (Ruelland et al., 2002). The cell culture system allows proper and rapid phosphate uptake and enables homogenous treatments of cells with inhibitors. To determine the amount of PtdOH produced via PLC/DGK, short-term [33P]-orthophosphate labeling (15 min) was performed before cold exposure. As shown in Figs 5(a) and S2(a), PtdOH was rapidly synthesized (after 1 min) by the PLC/DGK-dependent pathway upon chilling, as previously reported. Pretreatment in the presence of 1 mM cPTIO before chilling did not impair PLC-dependent PtdOH production (Fig. 5a). Similarly, we measured the production of PtdOH via the PLD pathway after long-term [33P]-orthophosphate labeling (16 h), which allows phosphate incorporation into membrane phospholipids, followed by a 15-min chase with unlabeled phosphate. As for PLC/DGK activity, the PtdOH produced via the PLD pathway was detected within the first 1 min of cold exposure (Figs 5b and S2b). Nevertheless, cPTIO treatment did not modify the production of PtdOH by the PLD pathway (Fig. 5b). These results indicate that the production of PtdOH in response to low temperature is not regulated by NO endogenously evoked upon cold stress.

image

Figure 5.  The nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) does not affect cold-induced phosphatidic acid (PtdOH) synthesis in Arabidopsis thaliana. (a) Quantification of phospholipase C (PLC)/diacyglycerol kinase (DGK)-PtdOH concentrations upon cold exposure, in the absence (open symbols) or presence (closed symbols) of 1 mM cPTIO. Total lipids were labeled with [33P]-orthophosphate for 15 min and extracted as described in the Materials and Methods section. Lipids were subsequently developed on thin-layer chromatography (TLC) using the SS1 solvent system. Values were normalized by reference to phosphatidylinositol and phosphatidylcholine (PtdIns + PtdCho) abundance. (b) Quantification of phospholipase D (PLD)-PtdOH concentrations upon cold exposure, in the absence (open symbols) or presence (closed symbols) of 1 mM cPTIO. Total lipids were labeled with [33P]-orthophosphate for 16 h and extracted, following a pulse-chase with unlabeled phosphate, as described in the Materials and Methods section. Lipids were subsequently developed on TLC using the SS1 solvent system. Values were normalized by reference to PtdIns + PtdCho abundance.

Download figure to PowerPoint

NO regulates phosphosphingolipid concentrations during the cold stress response

In addition to PtdOH formation, we recently showed that cold stress triggers the rapid and transient synthesis of PHS-P and Cer-P (C. Dutilleul & I. Guillas, unpublished data). We therefore examined the possibility of crosstalk between NO and sphingolipid signaling during the cold stress response. We investigated the effect of modulating NO concentration on the synthesis of PHS-P and Cer-P in plant cell cultures exposed to low temperatures. As shown in Figs 6(a) and S3(a), cell treatment with 1 mM cPTIO before cold treatment did not affect the phosphosphingolipid pattern in unstressed cells. However, whereas most phosphorylated bands remained unchanged, treatment with the NO scavenger significantly increased PHS-P and Cer-P concentrations after cold exposure (+63% and +40%, respectively, after 5 min; < 0.01; Fig. 6a). The effect of cPTIO was observed at all the time-points tested. As PHS-P and Cer-P concentrations reached a plateau after 5 min of cold exposure, we selected this short-term treatment for subsequent experiments to prevent pleiotropic effects of the chemical tested. When different cPTIO concentrations were tested, a maximum stimulatory effect was observed for high concentrations (2 mM) for PHS-P and low concentrations (0.1 mM) for Cer-P, which may indicate different regulatory mechanisms for the phosphorylation of the two molecules (Fig. 6b). As cold-induced NO production is dependent upon NR activity, we examined the effect of the NR inhibitor tungstate on PHS-P and Cer-P concentrations. The concentrations of PHS-P and Cer-P were strongly increased in cells treated with 1 mM tungstate before cold exposure (+161% and +82%, respectively; Fig. 6c), to a higher level than that observed with cPTIO. To further establish the relationship between NO concentration and cold-induced PHS-P and Cer-P formation, exogenous treatments with GSNO, a natural NO-generating molecule in animal cells, were performed. The release of NO from GSNO also generates glutathione (GSH), which may itself affect cellular processes. Nevertheless, cell treatments with GSH up to 2 mM did not affect PHS-P or Cer-P concentrations following chilling (Fig. S4). In contrast, treatments of cultured cells with 1 mM GSNO before cold treatment led to a decrease in the PHS-P and Cer-P concentrations measured following cold exposure (37% and 55% decreases, respectively; Figs 7a and S3b). The inhibitory effect of GSNO treatment was particularly strong after a short cold treatment (up to 10 min) and decreased slightly thereafter (Fig. 7a). When analyzed after 5 min of cold exposure, the inhibition of PHS-P and Cer-P formation by GSNO showed a marked dose-dependence (Fig. 7b). Cer-P formation was strongly inhibited even at low doses (0.1 mM) of GSNO, with maximum inhibition from a concentration of 0.5 mM (55%; Fig. 7b). The inhibition of PHS-P formation increased together with GSNO concentration (up to 56% for 2 mM GSNO; Fig. 7b). We examined the effect of a series of NO donors previously used in plants (Fig. 7c). PHS-P and Cer-P synthesis triggered by cold exposure was strongly inhibited by DEA NONOate (89% and 92% inhibition for PHS-P and Cer-P, respectively), only slightly affected by SNAP (25% inhibition for Cer-P and no significant inhibition for PHS-P) and not significantly modified by SNP. To establish that the modulation of NO concentration during the cold response could also lead to a modification of phosphosphingolipid signal formation in planta, we performed phosphosphingolipid labeling and compared PHS-P and Cer-P formation in control and chilled WT, nia1nia2 and H7 plants (Fig. 8). To allow reproducible and efficient phosphate uptake in plant tissues, we used young plantlets (2 wk old) that were directly immersed in the labeling medium. [33P]-orthophosphate incorporation into sphingolipids was nevertheless lower in plantlets than in cultured cells and only two labeled phosphosphingolipids could be visualized in unstressed and stressed material (Fig. S5). From co-migration with labeled phosphosphingolipids extracted from cultured cells, we could establish that they corresponded to PHS-P and Cer-P (data not shown). As in cultured cells, exposure of WT plantlets to cold for 5 min led to an increase in both PHS-P and Cer-P concentrations (Fig. 8). No significant difference in PHS-P and Cer-P concentrations was observed in unstressed WT, nia1nia2 and H7 plantlets (Fig. S5). By contrast, PHS-P and Cer-P concentrations were higher in nia1nia2 and H7 plantlets after cold exposure compared with chilled WT plantlets (Fig. 8). Cer-P concentrations were increased by 32 and 41% in nia1nia2 and H7 plantlets, respectively (Fig. 8). PHS-P appeared to be even more strongly stimulated (184% and 59% for nia1nia2 and H7, respectively). Taken together, these data establish that NO is a regulator of PHS-P and Cer-P synthesis in chilled plants.

image

Figure 6.  The nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) stimulates cold-induced sphingolipid phosphorylation in Arabidopsis thaliana. (a) Quantification of the variation of phytosphingosine phosphate (PHS-P) (squares) and ceramide phosphate (Cer-P) (triangles) concentrations upon cold exposure, in the absence (open symbols) or presence (closed symbols) of 1 mM cPTIO. Phosphosphingolipids were labeled and extracted as described in the Materials and Methods section. Sphingolipids were subsequently developed on thin-layer chromatography (TLC) using the SS2 solvent system. Values represent the ratio of phosphosphingolipid (PS) abundance between stressed (4°C) and unstressed (22°C) cultured cells. (b) Dose-dependent stimulation of cold-responsive PHS-P (dark bars) and Cer-P (white bars) synthesis by cPTIO. The ratio of PS abundance between stressed (4°C) and unstressed (22°C) cultured cells was determined after 5 min of cold exposure. Values were normalized by reference to cells treated with water (0). PHS-P and Cer-P increases (mean ± SE) after cold treatment in the control experiments were 2.5 ± 0.33 and 2.9 ± 0.22 fold, respectively. (c) Effect of tungstate on cold-responsive PHS-1 (closed bars) and Cer-P (open bars) synthesis. Tungstate and cPTIO were used at 1 mM. The ratio of PS abundance between stressed (4°C) and unstressed (22°C) cultured cells was determined after 5 min of cold exposure. Values were normalized by reference to cells treated with water (control). PHS-P and Cer-P increases (mean ± SE) after cold treatment in the control experiments were 2.8 ± 0.25 and 2.7 ± 0.12 fold, respectively.

Download figure to PowerPoint

image

Figure 7.  The nitric oxide (NO) donor nitroso-gluthatione (GSNO) inhibits cold-induced sphingolipid phosphorylation in Arabidopsis thaliana. (a) Quantification of the variation of phytosphingosine phosphate (PHS-P) (squares) and ceramide phosphate (Cer-P) (triangles) concentrations upon cold exposure, in the absence (open symbols) or presence (closed symbols) of 1 mM GSNO. Phosphosphingolipids were labeled and extracted as described in the Materials and Methods section. Sphingolipids were subsequently developed on thin-layer chromatography (TLC) using the SS2 solvent system. Values represent the ratio of phosphosphingolipid (PS) abundance between stressed (4°C) and unstressed (22°C) cultured cells. (b) Dose-dependent stimulation of cold-responsive PHS-P (closed bars) and Cer-P (open bars) synthesis by GSNO. The ratio of PS abundance between stressed (4°C) and unstressed (22°C) cultured cells was determined after 5 min of cold exposure. Values were normalized by reference to cells treated with water (0). PHS-P and Cer-P increases (mean ± SE) after cold treatment in the control experiments were 2.7 ± 0.28 and 2.6 ± 0.18 fold, respectively. (c) Effect of S-nitroso-N-acetyl-D,L-penicillamine (SNAP), 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA NONOate) and sodium nitroprusside (SNP) on cold-responsive PHS-P (closed bars) and Cer-P (open bars) synthesis. All donors were used at 1 mM. The ratio of PS abundance between stressed (4°C) and unstressed (22°C) cultured cells was determined after 5 min of cold exposure. Values were normalized by reference to cells treated with water (control). PHS-P and Cer-P increases (mean ± SE) after cold treatment in the control experiments were 2.7 ± 0.31 and 2.8 ± 0.27 fold, respectively.

Download figure to PowerPoint

image

Figure 8.  Nitric oxide (NO) depletion stimulates cold-responsive sphingolipid phosphorylation in planta. Quantification of the variations of phytosphingosine phosphate (PHS-P) (closed bars) and ceramide phosphate (Cer-P) (open bars) concentrations in Arabidopsis thaliana wild-type (WT), nia1nia2 and H7 plantlets exposed to cold. Phosphosphingolipids were labeled and extracted as described in the Materials and Methods section. Sphingolipids were subsequently developed on thin-layer chromatography (TLC) using the SS2 solvent system. The ratio of phosphosphingolipid (PS) abundance between stressed (4°C) and unstressed (22°C) plantlets was determined after 5 min of cold exposure. Values were normalized by reference to WT plantlets (Col-0). Error bars are +SE.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The importance of NO for plant responses to fluctuating environmental conditions has been illustrated during the last decade (Besson-Bard et al., 2008; Neill et al., 2008). In the present paper, we show that NO is produced in chilled A. thaliana and functions as a signal in the early cold transduction network.

Using the fluorescent dye DAF2-DA for NO detection, we observed that NO was produced during the first few hours following cold exposure, much more rapidly than previously reported (48 h in Corpas et al., 2008; 24 h to 14 d in Zhao et al., 2009). As this was confirmed by chemiluminescence measurements on whole plants, artefactual production resulting from the experimental procedure can be ruled out. Moreover, the ability of the NO scavenger cPTIO to interfere with fluorescence detection clearly establishes that the fluorescence detected reflects bona fide NO detection. NO was not detected in chilled leaves of the NR mutant nia1nia2 or in tungstate-treated plants. As the nia1nia2 mutant also showed a low arginine content, Modolo et al. (2006) proposed that the lower NO production in nia1nia2 may also be attributable to the impairment of arginine-dependent mechanisms. Together with the absence of inhibition by the NO synthase inhibitor l-NAME, our data indicate that the rapid NO production is mainly dependent upon NR activity and not on an nitric oxide synthase (NOS)-like activity. This hypothesis is compatible with the slight increase in the nitrite content observed in chilled leaves. We also found that the NO produced was efficiently dissipated in plants over-expressing the nonsymbiotic hemoglobin AHb1. Previous reports associated AHb1 function with NO turnover, in particular in plants experiencing oxygen deprivation (Perazzolli et al., 2004). Whether AHb1 is operating in planta to control NO bioactivy during cold stress is currently unknown. Nevertheless, we and others observed that AHb1 transcripts accumulate in chilled plants (Shimoda et al., 2005; Sasakura et al., 2006). As cold-responsive AHb1 induction is impaired by cPTIO, cold-induced NO could serve as a signal to induce AHb1 expression and therefore modulate its own bioactivity.

A major response of plants to chilling is the activation of the CBF-dependent pathway which regulates the expression of c. 100 COR genes (referred to as the CBF regulon) (Van Buskirk & Thomashow, 2006). We showed that the expression of two CBF transcription factors, CBF1 and to a lesser extent CBF3, was greatly reduced in chilled nia1nia2 and H7 plants compared with WT plants. Similar results were obtained in WT plants infiltrated with cPTIO before cold exposure (data not shown). By contrast, the transcript level for CBF2 was not affected by NO depletion. These data are in accordance with previous reports suggesting different regulation and/or functions for CBF2 compared with CBF1/3 (Novillo et al., 2004, 2007). Consistent with the decrease in CBF1/3 expression, the cold induction of members of the CBF regulon such as LTI30, LTI78 and COR15a was also impaired in nia1nia2 and H7 plants. To our knowledge, these data constitute the first evidence linking NO signaling with the CBF-dependent pathway. However, the expression of ZAT12, another cold-responsive transcription factor, was not modified in nia1nia2 and H7 mutants. These data contrast with previous reports on ZAT12 regulation using treatments with NO gas or NO donors, and may reflect different effects of exogenously applied and naturally induced NO (Parani et al., 2004; Palmieri et al., 2008).

Among the most rapid responses of plants to chilling is the generation of PtdOH from the activity of both the PLD and PLC/DGK pathways (Ruelland et al., 2002). Several studies have linked PtdOH production to NO, which would act upstream of the PLD and/or PLC/DGK pathways to trigger PtdOH synthesis (Laxalt et al., 2007; Distefano et al., 2008; Lanteri et al., 2008). Nevertheless, the depletion of NO by cPTIO had no effect on the synthesis of PtdOH by either the PLC/DGK or the PLD pathway after cold exposure. Although our results show that NO is not acting upstream of PtdOH synthesis, we cannot exclude the possibility that it participates in cold transduction downstream of the PtdOH production, as recently observed for the PtdOH-mediated production of NO triggered by extracellular ATP in tomato cells (Sueldo et al., 2010).

We recently found that sphingolipid-derived signals (e.g. a long-chain base phosphate, PHS-P and a Cer-P) are also rapidly and transiently generated in response to cold in A. thaliana (C. Dutilleul & I. Guillas, unpublished data). In the present paper, we provide evidence that PHS-P and Cer-P formation is negatively regulated by the endogenous NO induced during the cold response. First, PHS-P and Cer-P concentrations were higher when NO concentrations were decreased either by cPTIO treatment or by genetic means in nia1nia2 and H7 plants. As serine is a precursor of sphingolipids, the effect observed in the nia1nia2 mutant could be the consequence of an altered nitrogen metabolism. However, this is unlikely as the nia1nia2 mutant has a higher serine content than WT plants (Modolo et al., 2006). Moreover, the effects of modifying NO concentrations via cPTIO or by over-expressing AHb1, which reacts directly with NO, also suggest a direct link between NO and LCB-P/Cer-P formation. However, increasing the NO concentration using NO donors greatly impaired LCB-P/Cer-P formation. In the absence of pharmacological treatment, PHS-P and Cer-P are formed despite the production of NO during cold stress, which suggests a partial or controlled effect of NO inhibition in physiological conditions. These findings establish the first link between sphingolipids and NO signaling in plants. How NO affects PHS-P and Cer-P formation in chilled plants is currently unknown. Nevertheless, modifications of the pattern of nitrated and nitrosylated proteins following cold exposure have been recently reported in pea (Pisum sativum) and Brassica juncea (Corpas et al., 2008; Abat & Deswal, 2009). Direct regulation of sphingolipid kinases or phosphatase by S-nitrosylation or tyrosine nitration in cold-stressed plants is therefore possible, and enzymatic studies will be required to evaluate this hypothesis.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Dr I. Wilson (University of Bristol, UK) and Prof. M. Delledonne (University of Verona, Italy) for providing us with the nia1nia2 mutant line and the AHb1-over-expressing line, respectively. This work was supported by the Agence Nationale de la Recherche (ANR) (grant BLAN 071_184783), CNRS and Université Pierre et Marie Curie-Paris 6.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Effect of the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) on cold-regulated gene expression in chilled Arabidopsis thaliana Columbia (Col-0) leaves.

Fig. S2 Thin-layer chromatography (TLC) patterns of labeled phospholipids extracted from cell cultures treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO).

Fig. S3 Thin-layer chromatography (TLC) patterns of labeled phosphosphingolipids extracted from cell cultures treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or nitroso-gluthatione (GSNO).

Fig. S4 Effect of glutathione (GSH) treament on cold-induced phytosphingosine phosphate (PHS-P) and ceramide phosphate (Cer-P) synthesis.

Fig. S5 Thin-layer chromatography (TLC) patterns of labeled phosphosphingolipids of wild-type, nia1nia2 and H7 plantlets subjected or not subjected to chilling.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

FilenameFormatSizeDescription
NPH_3500_sm_FigS1.ppt118KSupporting info item
NPH_3500_sm_FigS2.ppt484KSupporting info item
NPH_3500_sm_FigS3.ppt767KSupporting info item
NPH_3500_sm_FigS4.ppt138KSupporting info item
NPH_3500_sm_FigS5.ppt526KSupporting info item
NPH_3500_sm_SupportingInformationLegends.doc28KSupporting info item