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Keywords:

  • reticulata;
  • chloroplast;
  • genetics;
  • leaf organogenesis;
  • oxidative stress;
  • reactive oxygen species;
  • stomata

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Genetically tractable model plants offer the possibility of defining the plant O3 response at the molecular level. To this end, we have isolated a collection of ozone (O3)-sensitive mutants of Arabidopsis thaliana. Mutant phenotypes and genetics were characterized. Additionally, parameters associated with O3 sensitivity were analysed, including stomatal conductance, sensitivity to and accumulation of reactive oxygen species, antioxidants, stress gene-expression and the accumulation of stress hormones. Each mutant has a unique phenotypic profile, with O3 sensitivity caused by a unique set of alterations in these systems. O3 sensitivity in these mutants is not caused by gross deficiencies in the antioxidant pathways tested here. The rcd3 mutant exhibits misregulated stomata. All mutants exhibited changes in stress hormones consistent with the known hormonal roles in defence and cell death regulation. One mutant, dubbed re-8, is an allele of the classic leaf development mutant reticulata and exhibits phenotypes dependent on light conditions. This study shows that O3 sensitivity can be determined by deficiencies in multiple interacting plant systems and provides genetic evidence linking these systems.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Plant response to the air pollutant ozone (O3) is complex and involves many biological processes at several levels. O3acts in the apoplast and requires access to this compartment via stomata. Thus, stomatal aperture determines the effective O3 dose seen by a given plant (Kollist et al. 2007). Intriguingly, stomata are also the first line of defence against some types of pathogens. Stomatal regulation is integrated with innate immunity-signalling pathways (Melotto et al. 2006) via connections in reactive oxygen species (ROS), salicylic acid (SA) and nitric oxide signalling, processes that are also central to the O3 response. Once in the leaf, O3 rapidly reacts with components of the cell wall, apoplastic fluids and plasma membrane (Kangasjärvi et al. 1994). O3 breaks down to form other ROS, such as H2O2, superoxide anion (O2•−) and singlet oxygen (Kangasjärvi et al. 1994). In turn, these ROS induce the active production of further ROS by the plant itself, termed the oxidative burst (Wohlgemuth et al. 2002). These direct and indirect O3-induced ROS signals then trigger downstream responses. Antioxidant pathways have an important role of protecting against ROS damage and modulating ROS signals (Langebartels et al. 2000). Most notable among these systems are ascorbic acid, glutathione and the enzymes of the Halliwell–Asada–Foyer pathway, such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). The presence of ascorbic acid in the apoplast is an important ROS scavenger able to diminish the effects of O3, especially early in the exposure (Conklin & Barth 2004). The perception of O3-induced oxidative stress results in the reprogramming of transcription (Broschéet al. 2007). Plant stress hormones play an integral role in the O3 response. SA, jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) are important regulators of O3 responses at several different levels (Overmyer, Brosché & Kangasjärvi 2003). All of these hormones induce defence responses. SA and ET promote lesion formation, and JA is involved in lesion containment (Overmyer et al. 2000; Tuominen et al. 2004). ABA is involved in the regulation of stomata (Kangasjärvi, Jaspers & Kollist 2005). The complex interaction of these signals is required for an effective defence response and, when out of balance, determines the extent of lesion development (Overmyer et al. 2003).

Physiological, stomatal, biochemical, antioxidant, gene expression and stress hormone signalling responses are all integrated to produce the various effects observed in plants subjected to O3 or other oxidative stresses. Largely, these various responses have been studied in isolation with only one or a few of these systems under study at a time. There are examples of more comprehensive papers: a study of six sensitive/tolerant pairs of clones, cultivars or populations of plants demonstrated that of the eight parameters investigated, only increased ET evolution provided a consistent explanation for sensitivity or tolerance (Wellburn & Wellburn 1996). Puckette, Weng & Mahalingam (2007) have studied the O3 sensitivity of 38 accessions of the model legume Medicago by assaying a large number of antioxidant parameters. Furthermore, the accumulated body of work with the tobacco O3-sensitive (BelW3) and -tolerant (BelB) pair (Heggestad 1991) has contributed to our understanding of O3 responses, especially at the biochemical level. However, all of these studies are limited by the complex genetic heterogeneity between the sensitive/tolerant cultivars or lines.

The application of genetics, through the use of genetically tractable model systems, like Arabidopsis thaliana, can be used to identify new links at the molecular level in the plant response to O3. Forward genetic screens for O3-sensitive Arabidopsis mutants have been used by several groups and have made a significant contribution to plant biology. The radical-induced cell death (rcd1) mutant, isolated in our lab, is sensitive to O3 and extracellular O2•− but not H2O2 (Overmyer et al. 2000). RCD1 encodes a novel protein (Ahlfors et al. 2004) that has been shown to interact with a number of nuclear transcription factors (Belles-Boix et al. 2000). Upon stress treatment, RCD1 is localized also outside of the nucleus and interacts with proteins at the plasma membrane (Katiyar-Agarwal et al. 2006). RCD1 defines a small gene family whose members are also involved in stress responses (Borsani et al. 2005). Together, these studies indicate that RCD1 plays a central role in regulating the response to multiple stresses involving ROS. In another screen, the O3-sensitive jasmonate-insensitive (oji) mutant has strengthened our understanding of the role of jasmonate signalling in O3 response (Kanna et al. 2003). Finally, a subgroup of mutants from the sensitive to O3 (soz) screen was renamed to vitamin c (vtc) when it was discovered that they were ascorbate (AA) deficient (Conklin, Williams & Last 1996; Conklin et al. 2000). This work has resulted in the clarification of the role of AA in plant responses to O3 and other stresses. Importantly, these mutants have facilitated definition of plant AA biosynthesis pathways (Smirnoff, Conklin & Loewus 2001). Taken together, these O3-sensitive mutants demonstrate the power of forward genetics in resolving the complex molecular pathways and networks involved in oxidative stress response.

As illustrated by several recent reviews (Overmyer et al. 2003; Conklin & Barth 2004; Kangasjärvi et al. 2005), significant progress has been made at many levels of O3 research. However, O3 and other oxidative stress responses remain inadequately defined at the molecular level, and it is likely that novel factors involved in plant O3 response remain unknown. Further research is required to identify new players and to link seemingly unrelated plant systems. In this paper, we describe the further application of forward genetics to questions of O3 response. We have isolated a collection of additional O3-sensitive mutants based on the appearance of O3-induced cell death lesions in the O3-tolerant Columbia-0 (Col-0) accession of A. thaliana. Multiple parameters, previously known to be associated with O3 sensitivity, were analysed in order to assess the basis and complexity of O3 sensitivity.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Mutant screening and genetic mapping

Arabidopsis thaliana ecotype Col-0 seeds were mutagenized and screened for O3 sensitivity as described in Langebartels et al. (2000). Mutants were crossed to determine allelism. Populations derived from crosses with Col-0 and Landsberg erecta (Ler) were used for segregation and mapping studies, respectively. Linkage analyses of F2 recombinant populations were perfomed using cleaved amplified polymorphic sequence and microsatellite markers (http://www.tair.org).

Microscopy, chlorophyll measurements and detection of ROS

Cell density was quantified from images of second and third true leaves of 15-day-old plants taken with an inverted confocal microscope (Zeiss LSM510 META, Germany) using a 20 × 0.50 water objective. Fluorescence was excited at 543 nm with a HeNe diode laser, and detected with a 560-nm-long pass emission filter. For chlorophyll content measurements, leaf discs were extracted in 80% acetone with 0.01% MgCO3, and absorbance at 646 and 663 nm was measured with Shimadzu UV2100 (Shimadzu Corp., Kyoto, Japan) spectrophotometer. Accumulation of H2O2 was analysed by using 3,3'-diaminobenzidine 4 HCl (DAB) according to Schraudner et al. (1998). O2•− was detected by nitroblue tetrazolium (NBT) staining according to Wohlgemuth et al. (2002).

Treatments with O3, superoxide and hydrogen peroxide (H2O2)

Three-week-old plants were exposed to a 6 h pulse of O3 as described in Langebartels et al. (2000). The O3 concentrations used in experiments varied between 250 and 350 nL L−1. Because of the stochastic physiological nature of O3 response and variation in batches of plants, test exposures with control sensitive and tolerant (rcd1 and Col-0) plants were used to determine the concentration required for a typical and consistent response in a given experimental period. Times of measurement refer to hours after the onset of exposure. Clean-air controls refer to plants kept in ambient non-filtered indoor air with an O3 concentration of <10 nL L−1. To generate radicals in the apoplastic space, detached leaves were infiltrated either with superoxide (O2•−)-generating system xanthine and xanthine oxidase (XXO; 0.5 mm/0.05 unit mL−1; Sigma, St Louis, MO, USA) or with H2O2-generating system glucose and glucose oxidase (GGO; 2.5 mm/2.5–250 units mL−1; Calbiochem, San Diego, CA, USA) in 10 mm sodium phosphate buffer, pH 7.0 (Jabs, Dietrich & Dangl 1996). Cell death was quantified as ion leakage from rosette leaves into 18 MΩ water for 1 h, measured with conductivity meter (Mettler Toledo GmbH, Greifensee, Switzerland) and expressed as a % of total ions, quantified after boiling.

Stomatal conductance and antioxidant assays

Stomatal conductance was measured from the abaxial leaf side with a steady-state diffusion porometer (AP4; Delta-T, Cambridge, UK). Poromoter was calibrated with a calibration plate daily prior to measurements. Some of the key measurements were repeated using the adaxial leaf surface with similar results. APX and SOD activity determinations were performed as described in Marklund (1985) and Foyer, Dujardyn & Lemoine (1989), respectively. AA and dehydroascorbate were analysed by GC–MS as described (Wingsle & Moritz 1997). GSH was extracted in 0.1 m HCl by grinding a freeze-dried rosette in 1 mL of acid and incubating on ice for 30 min. Cleared supernatant was used to measure the GSH and GSSG contents by a derivatization method (Newton, Dorian & Fahey 1981; Creissen et al. 1999).

Plant hormones and gene expression

Ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) were measured as described (Langebartels et al. 2000). ABA, SA and JA were quantified with the vapour-phase extraction method described by Schmelz et al. (2003) using 100 ng of 13C1–SA, 50 ng of dihydrojasmonic acid and 20 ng of D6–ABA from Icon Isotopes (Summit, NJ, USA) as internal standard for each sample. GC–MS analysis was performed on a Trace-DSQ from Thermo (Sweden) as described previously (Brader et al. 2007). Expression of 74 defence-related genes was studied by a custom-made cDNA array analysis as described in Tuominen et al. (2004).

Statistical analysis

Data in all figures are presented as means ± SD. In Figs 2, 3 and 7, means marked by different letters are statistically significant (P < 0.01) by two-way analysis of variance plus Tukey's test. In Fig. 5, only rcd3 was significantly different from Col-0 by the same test. In all experiments, five plants were sampled (n = 5), except Fig. 7 (n = 4) and Table 2(n = 3). Each replicate (sample) represented a different individual plant, not the leaves from the same plant. All experiments were performed at least three times with similar results; representative results are shown.

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Figure 2. Chlorophyll concentration (a) and cell density (b) of 3-week-old re-8 and Col-0 wild-type plants grown under normal (250 µmol m−2 s−1) and low (30 µmol m−2 s−1) light intensity with a photoperiod 12/12 h (light/dark). In addition, chlorophyll content and cell density of plants grown under normal light but shorter day length (8/16) are shown. Phenotype development of all other mutants in this study was independent of photoperiod (not shown).

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Figure 3. Quantification of ozone (O3)- (a–c) and superoxide (O2•−) and H2O2- (d) induced leaf damage on the basis of relative ion leakage (% total ions). (a) Leaves from 3-week-old plants were collected after 250 nL L−1 O3 exposure for 6 h. (b) Same as (a) except that plants were grown and exposed to O3 under low light conditions (30 µmol m−2 s−1). (c) Same as (b) except that light conditions were normal (150 µmol m−2 s−1) during O3 exposure. To test the effect, O2•− and H2O2, leaves from clean-air-grown plants were infiltrated with xanthine and xanthine oxidase or glucose and glucose oxidase, respectively. Ion leakage was measured 24 h after the infiltration.

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Figure 7. Ozone (O3)-induced stress hormone accumulation. The accumulation of the hormones, (a) salicylic acid, (b) jasmonic acid and (c) abscisic acid, was determined at 0, 1.5, 3 and 8 h after the beginning of a 6 h pulse of 250 nL L−1 O3 in Col-0 and O3-sensitive mutant plants.

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Figure 5. Leaf stomatal conductance of re-8, rcd3, rcd4 rcd6 and Col-0 wild type exposed to 250 nL L−1 of ozone (O3) for 6 h.

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Table 2.  Basal antioxidants of ozone (O3)-sensitive mutantsa
GenotypeAscobic acid (µg g−1 FW)Glutathione (µmol g−1 DW)APX activity (U mg−1 prot.)SOD activity (U mg−1 prot.)
  • a

    The basal concentration of total ascorbate (AA), total glutathione and the basal activities of ascorbate peroxidase (APX) and superoxide dismutase (SOD) were determined in re-8, rcd3, rcd4 rcd6 and Col-0 wild type from 3-week-old plants grown in clean-air control conditions. None of the mutants exhibited significant differences from Col-0. Means are expressed ± SD and are not statistically different (P > 0.1) by analysis of variance.

Col-0734.5 ± 82.13.69 ± 2.250.051 ± 0.0150.280 ± 0.145
re-8880.1 ± 51.93.42 ± 1.860.039 ± 0.0170.330 ± 0.047
rcd3771.6 ± 77.53.32 ± 2.110.029 ± 0.0080.221 ± 0.075
rcd4 rcd6667.1 ± 65.82.65 ± 1.570.039 ± 0.0090.171 ± 0.025

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Genetic characterization of O3-sensitive radical-induced cell death (rcd) mutants

We have isolated a series of O3-sensitive mutants (Overmyer et al. 2000), including a new allele of a mutant isolated previously in other screens (Barth & Conklin 2003; González-Bayón et al. 2006). These mutants, which each have their own characteristic pattern of O3-induced damage (Fig. 1), were assigned the designation rcd2 through rcd4.

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Figure 1. Ozone (O3)-induced damage in Col-0, re-8, rcd3, rcd4 rcd6. Three-week-old plants were exposed to 250 nL L−1 for 6 h. Photos were taken at 24 h. Collapsed tissue on rcd4 rcd6 is indicated with a black arrow.

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The mode of inheritance was determined by back-crossing to the parental Col-0. All mutants exhibited wild-type phenotypes in the F1 generation. Segregation ratios in the F2 generation and models of heritability are presented in Table 1a. The F2 segregation of rcd4 indicated that its O3 sensitivity was the result of two independent recessive mutations. The observed 1:3:7:21 ratio is variation of the 1:3:3:9 model. It is 4:12 (i.e. 1:3) segregation superimposed upon a 1:3:3:9 (1:3:4 + 3:12 + 9 = 1:3:7:21). This is the result of a cross where one locus is a heterozygote in a double mutant with two recessive loci. Thus, it is referred to as rcd4 rcd6 double mutant. While the segregation ratios observed are consistent with two recessive loci, because of the complexity of this segregation pattern, other models may still be possible. In isolation, the rcd4 and rcd6 single mutants have only subtle O3-sensitive phenotypes; leaf wrinkling in rcd4 and slight chlorosis in rcd6. Because of the weak single mutant phenotypes, all subsequent experiments used the homozygote rcd4 rcd6 double mutant. Genetic complementation tests indicated that these mutants all represent independent loci (Table 1b). The rcd2 mutant was mapped to BAC T28M21 on the bottom of chromosome II, which coincided with the localization of the phenotypically similar mutant lower cell density1-1 (lcd1-1) (Barth & Conklin 2003). Lack of genetic complementation in >20 F1 plants of rcd2 × lcd1-1 indicated that rcd2 is allelic to lcd1-1, which was subsequently shown to be an allele of reticulata (re) (González-Bayón et al. 2006). Sequencing of At2g37860 revealed that rcd2 has single G to A transition at the splice site of the fifth intron that results in a predicted truncated protein. Therefore, the rcd2 mutant is referred to as reticulata-8 (re-8). The rcd3 mutant has been mapped between markers SNP132 and SGCSNP9742 in chromosome I. The rcd4 rcd6 mutant loci have been mapped to chromosome 1 showing 12.96 ± 1.96% recombination to the marker ciw12 and to the bottom of chromosome 2 showing 12.50 ± 2.08% recombination to marker athBIO2.

Table 1.  Genetics of the rcd mutants: (a) inheritance of the ozone (O3)-sensitive phenotypes showing segregation in the F2 generation of a back-cross between the respective mutants and their wild-type Col-0 parental background; all crosses were performed using the mutant plant as the pollen donor, and (b) mutant allelism (genetic complementation) tests
(a)
MutantF2 segregationRatio/ModelX2P =
re-861/209a1:3/single recessive0.7630.382
rcd372/218a1:3/single recessive0.0040.946
rcd4 rcd68/33/69/194b1:3:7:21c/two recessive1.190.754
(b)
Crossd03 Sensivity (sensitive/total tested)
  • Reported is the number of O3-sensitive individuals divided by the total number of individuals tested. Plants tested were the F1 progeny of cross between the O3-sensitive mutants as indicated. Crosses were done as listed above (acceptor × donor).

  • a

    Two phenotype classes are reported as the number of plants scored as sensitive (lesion bearing)/tolerant.

  • b

    Four different phenotype classes are reported as the number of plants scored as sensitive/intermediate sensitive type 1/intermediate sensitive type 2/tolerant. Plants of the intermediate sensitive classifications did not exhibit lesion per se. Intermediate type 1 plants were characterized by leaf wrinkling, and intermediate type 2 by chlorotic spots.

  • c

    The ratio 1:3:7:21 is variation of the 1:3:3:9 model. It is 4:12 (i.e. 1:3) segregation superimposed upon a 1:3:3:9 (1:3:4 + 3:12 + 9 = 1:3:7:21). This is the result of a cross where one locus is a heterozygote in a double mutant with two recessive loci.

  • d

    Crosses are listed here in the order: pollen acceptor × pollen donor.

rcd3 × rcd4 rcd64/69
re-8 × rcd4 rcd61/38
rcd3 × re-80/8

Clean-air phenotypes of rcd mutants

Both visual and microscopic examinations of leaves of the mutants revealed that rcd4 rcd6 shows no morphological difference to Col-0 wild type, while rcd3 has slightly broader leaves. In contrast, re-8 clearly differs from wild type. The characteristic visual phenotype of re-8 is a reticulate pattern of pale coloration (Fig. 1). We measured chlorophyll content and cell density of re-8 and Col-0 plants grown under various light conditions. Mutant re-8 plants had significantly lower chlorophyll content (Fig. 2a) and reduced cell density in the palisade parenchyma (Fig. 2b) when grown under standard light intensity [photosynthetic photon flux density (PPFD), 250 µmol m−2 s−1] with a photoperiod of 12/12 h (light/dark). However, these phenotypes were absent when plants were grown under low light (<30 µmol m−2 s−1; Fig. 2a,b). Furthermore, when re-8 was grown under short-day length (8/16 h light/dark), it was visually indistinguishable from Col-0 and had the same chlorophyll content. Furthermore, the cell density in plants grown under normal (12/12 h light/dark) day with different light intensities (PPFD between 30 and 250 µmol m−2 s−1) did not differ between re-8 and Col-0 (Fig. 2b). Thus, the cell density phenotype of re-8 seems to be the result of photoperiod-specific light intensity-dependent processes affecting leaf development. The re-8 mutant is also temperature sensitive; it is stunted considerably when grown under low temperature. An additional allele, re-7 (SALK_037307), has phenotypes identical to re-8 (data not shown). No other mutants in this study exhibited light period dependency for phenotype development (data not shown).

Timing and pattern of O3-induced damage formation in mutants

The timing of O3-induced damage formation was similar for all mutants. Dark water-soaked lesions appeared at 12 h, they turned brown and collapsed and turned into dry lesions with distinct borders by 24 h. The pattern of affected tissue differed between mutants (Fig. 1). Lesions on re-8 were found only within the intervascular tissue. The rcd4 rcd6 mutant had randomly distributed lesions. In rcd3, damage was concentrated along the vascular bundles. Frequently, and especially at higher O3 concentrations (as shown in Fig. 4), these mutants can exhibit a second mode of damage formation where large areas of confluent lesions collapse entire tissues. In this case, visible damage was apparent as a region of turgor loss and tissue collapse as early as 3 h.

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Figure 4. Accumulation of hydrogen peroxide (H2O2), stained with diaminobenzidine in Col-0, re-8, rcd3 and rcd4 rcd6 plants grown under standard growth conditions (control) and after treatment with 300 nL L−1 ozone (O3) for 6 h. In addition, leaves from re-8 plants grown under low light intensity (30 µmol m−2 s−1) were stained (re-8 low light). Last panel illustrates typical O3-induced lesions of studied genotypes. At least 10 leaves per genotype were stained; representative results are shown.

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Increased sensitivity to O3 and superoxide, but not to H2O2

To quantify the observed O3 sensitivity and assess the response to O2•− and H2O2, we used ion leakage, an indicator of plasma membrane damage, as a measure of cell death. Consistent with their O3 sensitivity, all mutants exhibited ion leakage markedly higher than Col-0 after exposure to 250 nL L−1 O3 for 6 h (Fig. 3a).

As other re-8 phenotypes were light dependent, the O3 sensitivity of this mutant was also tested under varied light regimes. Growing plants under low light reduced stomatal conductance about twofold: conductance values for Col-0, re-8 and rcd1, respectively, were 311.1 ± 48.3, 369.4 ± 100.3, 460.6 ± 87.3 under normal light and 121 ± 38, 146 ± 30, 188 ± 57 under low light. To compensate for reduced O3 influx and maintain an equal effective O3 dose, the concentration of O3 was doubled to 500 nL L−1. Under these conditions, cell death was absent in re-8 and Col-0, while the O3-sensitive control, rcd1, still displayed its characteristic sensitivity (Fig. 3b). The presence of O3 lesions in rcd1 but not in re-8 supports the assertion that O3 tolerance in re-8 under low light was caused by a response to light intensity rather than reduced stomatal conductance. Accordingly, when re-8 and Col-0 plants were grown under low light intensity but treated with O3 under normal light intensity, significant cell death was evident in re-8 (Fig. 3c). This suggests the involvement of light-dependent processes as a factor causing O3 lesions in re-8.

O3 exerts its effect on plants via degradation into various ROS including O2•− and H2O2. To determine the ROS sensitivity of these mutants, plants were infiltrated with exogenous ROS-generating systems XXO for O2•−, and with GGO for H2O2 (Jabs et al. 1996; Alvarez et al. 1998), and ion leakage was measured at 12 h. XXO caused a marked increase in ion leakage in all plants. However, the increase was always significantly higher in mutants (Fig. 3d). Infiltration of leaves with GGO did not cause increased cell death (Fig. 3d). Direct treatment of leaves with various concentrations of H2O2 confirmed this result; mutants and Col-0 exhibited similar sensitivity to H2O2 over a wide range of concentrations (Supplementary Fig. S1). These results indicate that these O3-sensitive mutants are hypersensitive to O3 and O2•−, but not H2O2. However, we do not exclude the possibility that other ROS derived from O2•− or multiple ROS, such as O2•− and H2O2, could be required together for the observed ROS hypersensitivity.

Reactive oxygen species production in rcd mutants and Col-0

To elucidate the type and spatial distribution of ROS produced in rcd mutants, we infiltrated leaves with DAB or NBT, indicative of H2O2 and O2•−, respectively (Jabs et al. 1996; Thordal-Christensen et al. 1997). No DAB precipitate could be detected in most clean-air-grown plants (Fig. 4). Clean-air-grown re-8 displayed markedly higher DAB precipitation, which was always located around the vasculature (Fig. 4). Such DAB precipitate was absent in re-8 plants grown under low light (PPFD < 30 µmol m−2 s−1) and indicates that standard growth light of 250 µmol m−2 s−1 results in light-dependent perivascular H2O2 accumulation in re-8.

To analyse O3-induced production of ROS, plants were exposed to a standard O3 treatment, and leaves were sampled at 8 h (2 h after the 6 h treatment), excluding the detection of ROS from direct O3 breakdown products. The re-8 mutant displayed only somewhat higher O3-induced DAB precipitate around the vasculature (Fig. 4). Staining of O3-exposed re-8 with NBT revealed intense formation of dark blue precipitate at the borders of spreading lesions (Supplementary Fig. S2). This indicates that O2•− is the major ROS accumulating adjacent to expanding lesions in re-8. In contrast, rcd3, rcd4 rcd6 and Col-0 seemed to accumulate primarily H2O2 after O3 treatment (Fig. 4), and sites of accumulation coincided with the pattern of leaf injury in these genotypes. Although Col-0 is extremely tolerant to O3 and rarely forms visible damage, some spot-like injury can occur, especially at the higher end of our concentration range (≥300 nL L−1). Under these conditions, damage is always greater in the sensitive genotypes. In Col-0, H2O2 production occurs only with the appearance of such damage and is located on the borders of these spot-like lesions (Fig. 4). Similarly, H2O2 accumulation occurred in developing lesions and at the border of lesions in rcd3 and rcd4 rcd6. Notably higher deposition of NBT precipitate in O3-treated rcd3 and rcd4 rcd6 indicates that also O2•− accumulation was induced in these genotypes (data not shown). However, O2•− accumulation was randomly distributed, while H2O2 accumulation was more distinctly related with lesion formation in rcd3 and rcd4 rcd6.

Stomatal regulation

Measurements of stomatal conductance before, during (2 h, 4 h) and after (6 h) of 250 nL L−1 O3 exposure (Fig. 5) revealed some differences in stomatal behaviour. Col-0, re-8 and rcd4 rcd6 responded similarly to O3, closing their stomata by the end of the exposure. However, conductance was already significantly higher prior to O3 exposure in rcd3. Furthermore, rcd3 exhibited no O3-induced closure after 3 h and only a modest decrease by the end of the exposure. This indicates that, of the O3-sensitive mutants described here, altered stomatal behaviour accounts for O3 sensitivity only in rcd3.

O3 sensitivity of rcd mutants is not caused by reduced antioxidant capacity

To address whether O3 sensitivity of these mutants is caused by a deficiency in antioxidant capacity, such as that seen in soz/vtc mutants (Conklin et al. 1996), the core antioxidant pathway was analysed. Basal AA was similar in all mutants and wild type (Table 2). Concentrations of second major antioxidant, glutathione, were also similar in Col-0 and mutants (Table 2). Additionally, the basal activities of two major antioxidant enzymes APX and SOD were assayed and found not to significantly differ from wild-type Col-0 (Table 2). Taken together, this indicates that O3 sensitivity of these mutants is not likely caused by a gross inability to detoxify ROS within these systems.

O3-induced accumulation of stress hormones

We analysed O3-induced synthesis of the gaseous hormone ET and the levels of its immediate precursor ACC. Additionally, we determined the levels of three other stress hormones, SA, JA and ABA, in O3-exposed plants. O3 exposure resulted in the increased accumulation of all of these hormones to some extent in all genotypes. Generally, the hormone response was either faster or of higher magnitude in the O3-sensitive mutants (Figs 6 & 7).

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Figure 6. Ozone (O3)-induced synthesis of ethylene (ET) and its immediate precursor 1-aminocyclopropane-1-carboxylic acid (ACC) from the leaves of three O3-sensitive mutants (re-8, rcd3, rcd4 rcd6) and Col-0. ACC concentrations (a, b) and ET evolution (c, d) were measured from plants exposed to 250 nL L−1 O3 for 6 h (filled symbols) and to clean-air control (blank).

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All the observed changes of ACC and ET production were O3 specific as clean-air control plants show no changes (Fig. 6). O3 caused a rapid increase in ACC synthesis at 2 h in all genotypes studied. Importantly, this increase was significantly higher in the mutants than in Col-0 (Fig. 6a,b). In contrast to Col-0, in which ACC quickly returned to control levels, all mutants maintained high ACC concentrations until the 24 h time-point. Similarly, O3 triggered ET evolution in all genotypes (Fig. 6c,d). ET evolution lasted longer and was significantly higher in re-8 and rcd3 (Fig. 6c,d). SA accumulation was similar in all plants by 8 h post-fumigation. However, SA induction was higher in re-8 at 1.5 h and in all O3-sensitive mutants at 3 h (Fig. 7a). Low-level accumulation of JA was seen in O3-exposed Col-0 and rcd3 at time-points after 3 h. At 3 and 8 h, the O3-sensitive re-8 and rcd4 rcd6 accumulated high levels of JA (Fig. 7b). ABA levels were also induced by O3 in all plants; however, only at the latest time point of 8 h (Fig. 7c).

O3-induced gene expression

DNA macroarray hybridization was utilized to follow the expression of 74 selected stress-related and hormone signalling marker genes (for the complete data set and a list of genes’ AGI codes, see Supplementary Table S1 and Fig. S3) at 8 h post-O3 exposure and unexposed controls in Col-0 and the three O3-sensitive mutants. Expression of antioxidant genes was enhanced; however, largely similar to Col-0, indicating an intact transcriptional antioxidant response (Fig. 8a). O3-induced expression of these genes was somewhat higher in mutants, consistent with their heightened oxidative stress. One notable difference is the reduced basal level and lack of O3 induction of chloroplastic iron SOD in re-8 (Fig. 8a). Consistent with the induction of cell death by O3, pathogen response (PR) genes, especially those responsive to SA, are more highly expressed in mutants (Supplementary Fig. S3 and Table S1). Generally, markers for the O3-induced stress hormones, SA, JA, ET and ABA are all more highly expressed in the mutants (Supplementary Fig. S3 and Table S1). However, the O3 response of ABA marker genes (RAB18, ERD10, COR47) is weak in rcd4 rcd6 as compared with Col-0 (Fig. 8b), and the level of some JA-responsive genes (PDF1.2, LOX2, AOS) is depressed in re-8 under clean-air conditions (Fig. 8c).

image

Figure 8. Clean air and ozone (O3)-induced gene expression. The expression of select genes is depicted as fold difference to Col-0 according to the color-coded key at the bottom for the mutants re-8, rcd3 and rcd4 rcd6 (rcd4) under control clean air (CA) conditions and 8 h after the beginning of a 6 h × 250 ppb O3 exposure (8 h O3). Gene abbreviations are listed on the right and include a block of antioxidant genes (a) abscisic acid response genes (b), and jasmonic acid response genes (c). Full gene names and AGI codes can be found from Supplementary Table S1.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

We present here the characterization of three O3-sensitive rcd mutants. These mutants exhibit O3 sensitivity determined by multiple factors indicating that sensitivity is the result of the complex interplay of multiple systems. Some factors were common to all mutants, while others were unique to individual mutants.

AA is an important ROS scavenger involved in O3 responses, as demonstrated by the O3 sensitivity of AA-deficient mutants (Conklin et al. 1996). In order to test if the O3 sensitivity of rcd mutants could be explained by a gross deficiency in ROS detoxification, this study monitored the status of several key ROS detoxification systems, at the gene expression and anitioxidant metabolite levels. A large block of 12 antioxidant pathway genes was studied (Supplementary Fig. S2). These genes exhibit O3 responsiveness equal to or greater than that of wild-type plants (Supplementary Fig. S2), with one exception, FeSOD, as noted earlier. This indicates the transcriptional antioxidant response for these genes is intact and functional. No difference was found in AA levels, basal APX activity, basal total SOD activity and glutathione levels (Table 2). We conclude that ROS sensitivity of these mutants is not because of gross deficiencies in these key ROS detoxification systems.

All of the rcd mutants exhibited an oxidative burst; however, each mutant has its own unique pattern of ROS accumulation (Fig. 4), underscoring their mechanistic differences in damage formation. This study showed no correlation between damage (Fig. 3a) and the type or extent of ROS accumulation. Wohlgemuth et al. (2002) also observed a similar disjuncture between damage, site of production and reactive species produced in O3-exposed Arabidopsis. This observation suggests that a given ROS signal can summon different outcomes depending on the source, localization and context of other ROS signals in the cell. Accordingly, the rcd1 mutant is sensitive to apoplastic O2•− and O3 but more tolerant to chloroplast O2•− produced by paraquat (Ahlfors et al. 2004; Fujibe et al. 2004). In the Arabidopsis lsd1 mutant, which is a model for cell death regulation, ROS from NADPH oxidases RBOHD and RBOHF are required for the negative regulation of cell death (Torres, Jones & Dangl 2005). This underscores the multiple roles of ROS and the importance of the timing and location of their production. Taken together, these facts support the concept of an ‘ROS signature’ that has been previously suggested (Mahalingam & Fedoroff 2003). This refers to the fact that signals from different ROS sources that are spatially and temporally separated are integrated to determine a specific output.

The results in Figs 6 and 7 demonstrate that SA, JA and ET are induced by O3 exposure in these mutants, indicating that these mutants are not deficient in the biosynthesis of these hormones. These results are consistent with the previously suggested roles of plant stress hormones in the regulation of O3 defence and cell death responses (Overmyer et al. 2000, 2003). In several instances, SA and ACC/ET responses began prior to lesion appearance, suggesting that these are specific signalling events and not simply indicators of lesion formation. Significantly, Fig. 7c shows the induction of ABA in response to O3 treatment in Col-0 wild-type plants and to a greater extent in O3-sensitive mutants. In spite of the importance of ABA for stomatal regulation, and stomata for O3 response, O3-induced ABA accumulation has been largely neglected. Taken together, all these data suggest that hormone signalling imbalances are involved in the development of damage in these O3-sensitive mutants.

In addition to the mentioned features that were common to all sensitive genotypes, characteristics unique to their individual sensitivity mechanisms were revealed for each mutant.

The rcd4 rcd6 mutant phenotype requires two recessive mutations for full O3 sensitivity. The uncommon segregation ratio observed in rcd4 rcd6 was from segregation at one locus in a population from which one cross was done (Table 1). Individually, rcd4 and rcd6 are very minor O3 sensitivity loci that do not result in lesions. However, together in the double mutant, they exhibit a synergism resulting in tissue collapse. Altered ABA marker expression (Fig. 8b) suggests an impaired ABA response in the rcd4 rcd6 mutant. ABA biosynthesis appears intact (Fig. 7), suggesting insensitivity to ABA. ABA insensitivity is also a feature of the rcd1 mutant (Ahlfors et al. 2004).

We show that rcd2 is allelic to reticulata (re) and lcd1; this new allele results in a disrupted splice site in the last intron and predicts production of a truncated protein. González-Bayón et al. (2006) studied seven alleles of the classical genetic marker re and cloned the gene showing it was allelic to lcd1. The light-dependent phenotypes and predicted chloroplast localization of RE suggest that it is involved in chloroplastic processes, although chloroplast number and function are not affected in lcd1/re mutants (Barth & Conklin 2003; González-Bayón et al. 2006). The characteristic phenotype of re-8 is a pale reticulate phenotype when grown under standard Arabidopsis growth conditions (PPFD 250 µmol m−2 s−1 light, 12 h photoperiod; Fig. 1). Several studies (this study; Barth & Conklin 2003; González-Bayón et al. 2006) demonstrate that this was caused by lower cell density of leaf parenchyma in lcd1/re mutant plants, implicating RE in normal leaf development. Decreased mesophyll cell density is the primary defect in lcd1, and LCD1/RE is reported to control cell division early in the early stages of leaf development (Barth & Conklin 2003; Yu et al. 2007). Double mutant analysis with other variegated leaf mutants suggests that RE acts in a developmental pathway with CUE1 but not DOV1 (González-Bayón et al. 2006). Here, we show that re-8 developmental phenotypes are dependent on the combined effect of light intensity and photoperiod (Fig. 2). Furthermore, its O3 sensitivity is light intensity dependent (Fig. 3b,c). This suggests that RE is involved in processes driven by light dosage. Such dependence on environmental conditions for phenotype development has been seen previously in the variegated mutant immutans, whose bleaching is dependent on light levels, and variegated1, whose variegation pattern can be suppressed at temperatures below 20 °C (Yu et al. 2007). However, the photoperiod dependency seen here has not been previously reported in variegated mutants. RE together with At5g22790 comprise to a two-member gene family, encoding proteins of unknown function bearing predicted chloroplast targeting signals. It is likely that this gene pair is functionally redundant, as is often the case for genes involved in determining variegated phenotypes (Yu et al. 2007). Chloroplast envelope membrane localization has been experimentally confirmed for At5g22790 (Ferro et al. 2003), but not RE.

The depressed jasmonate-responsive gene expression in this mutant under non-stressed conditions suggests a role for jasmonates in both the developmental and O3 sensitivity phenotypes of this mutant.

Given the O3-sensitive phenotype and the increased vascular H2O2 accumulation of re-8 (Fig. 4), it is possible that RE is involved, at least indirectly, in chloroplast ROS signalling. Consistent with this idea, re-8 was unable to transcriptionally induce chloroplastic Fe SOD (Fig. 8a) in response to O3-induced oxidative stress. ROS signals are a common currency in the regulation of the chloroplast (Karpinski et al. 2003), and it has been shown that H2O2 from the chloroplast acts as a systemic stress acclimation signal (Karpinski et al. 1999). Importantly, light signalling has been linked to cell death control. Changes in light quality, perceived by the photosynthetic apparatus modulated cell death in the lsd1 mutant, a well-known cell death model (Mateo et al. 2004). Furthermore, perception of blue light by cryptochrome is required for execution of cell death triggered by the production of singlet oxygen (Danon, Coll & Apel 2006).

The rcd3 mutant has constitutively more open stomata and a lack of O3-induced stomatal closure (Fig. 5). Stomatal closure allows stress avoidance, and lack of this response may promote O3 damage in rcd3. The higher stomatal conductance in rcd3 together with its changes in stress hormone levels and signalling illustrate the complex way changes in multiple plant systems determine O3 sensitivity. Lesion propagation has been shown to be a hormone-dependent process, where SA and ET promote O3 lesion expansion. This study (Figs 5 & 6a) suggests that direct oxidative damage driven by higher initial or prolonged O3 influx in rcd3 may be the trigger of increased ET biosynthesis, which drives further lesion propagation. Thus, as has been suggested previously (Kangasjärvi et al. 2005), the level of O3 influx may determine lesion initiation.

Notably, O3-induced stomatal closure occurred in Col-0 plants already by 3 h; however, the accumulation of ABA first appears later at 8 h. This suggests a mechanism other than ABA is responsible for O3-induced closure, although other explanations, such as changes in ABA sensitivity, remain to be excluded. Interestingly, stomatal closure has recently emerged as a model for signal transduction, and H2O2 is an important signalling intermediate in this genetically defined pathway (Pei et al. 2000; Kwak et al. 2003). This suggests that O3-derived ROS may act directly in stomata to induce closure. Utilizing a new specialized apparatus, Kollist et al. (2007) have reported rapid stomatal closure within circa 10 min of O3 exposure. This rapid response was dependent on the class II protein phosphatase ABI2 and was apparently because of the direct perception of O3-derived ROS by stomata in Arabidopsis.

Work in our lab subsequent to this current study has identified RCD3 as At1g12480, which is expressed in guard cells and annotated as a distant homolog of fungal and bacterial dicarboxylate/malic acid transport proteins (Vahisalu et al. 2008). Electrophysiological studies indicate that RCD3 is required for slow (s-type) anion channel current. Taken together, this work contributes to our understanding, not only of O3 responses, but also basic signal transduction in the regulation of stomatal function.

O3-sensitive mutants provide genetic evidence that plant O3 response is under complex genetic regulation. The number of mutants isolated in this low/medium saturation screen implies that many loci are involved and suggests there are many O3-sensitive mutants that remain to be discovered. It is to be expected that O3 sensitivity can be conferred by deficiencies in a number of different systems. Importantly, this study shows that O3 sensitivity, within one genotype, can be determined by multiple factors, such as hormone responses, stomatal regulation, ROS accumulation, etc. This provides genetic evidence delineating previously unseen relationships between these factors. For example, in rcd3/slac1, it is likely that increased direct oxidative damage from unregulated O3 influx drives ET evolution, which further drives the accumulation of ROS. Beyond illustrating that point, this study shows that sensitivity screens can isolate novel mutants, and eventually their respective genes, which regulate a variety of known and novel O3-induced processes. Further work with O3-sensitive and other mutants of Arabidopsis should allow the definition, at the molecular level, of pathways and networks involved in oxidative stress response.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

We acknowledge the excellent technical help of Mrs. Baldeep Kular for the GSH determinations, Cheikh Diop for APX activity measurements and Thomas Moritz for AA determinations. GSH analyses were performed at the John Innes Centre in the lab of P.M. K.O. was supported by postdoctoral grants (decisions 202828 and 115034) from the Finnish Academy, and H.K. by Estonian Science Foundation (grant no. 6241) and by Targeted Funding Grant from Ministry of Research and Education (SF0180071507). Work in the lab of J.K. was supported by the Academy of Finland Centre of Excellence programmes 2000–2005 and 2006–2011, and by the Helsinki University Environmental Research Centre.

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  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Figure S1. Induction of cell death by hydrogen peroxide (H2O2). H2O2-induced cell death was quantified as ion leakage. Detached leaves were infiltrated with buffer containing various concentrations of H2O2. Leaf treatment was done in an excess volume of buffer (10 leaves in 50 mL) toensure that H2O2 was not cleared by leaf detoxification systems. H2O2 concentrations were monitored spectrophotometrically before and after leaf incubation, and never varied by more than 5–10%. Data are displayed as means (n = 5) with error bars representing SD.

Figure S2. Superoxide accumulation in re-8. Superoxide accumulation was visualized by nitro blue tetrazolium (NBT) staining. The reaction of NBT with superoxide results in the deposition of a dark purple formizan precipitate. Leaves were stained at 8 h after the beginning of a 6 h × 300 ppb ozone (O3) exposure. At least 10 leaves were stained in each of three independent exposures. Representative results are shown.

Figure S3. Clean air (CA) and ozone induced gene expression for all genes on the array. The expression is depicted as fold difference to Col-0 according to the colour coded key at the bottom for the mutants re-8, rcd3 and rcd4 rcd6 (rcd4) under control CA conditions and 8 h after the beginning of a 6 h × 250 ppb ozone exposure (8hO3). Gene abbreviations are listed on the right. The data including full gene names and AGI codes can be found from supplemental Table 1.

Table S1. Full array raw data set. The expression of 74 genes, selected for their involvement in stress and hormone signalling, were studied by macro array hybridization with samples from plants grown under control clean air conditions and 8 h after the begin of a 6 h (250 ppb) ozone exposure.

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