JXB Advance Access published online on September 5, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern222
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Genotypic variation in tolerance to elevated ozone in rice: dissection of distinct genetic factors linked to tolerance mechanisms
Japan International Research Center for Agricultural Sciences (JIRCAS), Crop Production and Environment Division, Abiotic Stress Tolerance Group, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan
* To whom correspondence should be addressed: E-mail: wissuwa{at}affrc.go.jp
Received 23 June 2008; Revised 28 July 2008 Accepted 4 August 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
|---|
Tropospheric ozone concentrations are increasing in many Asian countries and are expected to reach levels that adversely affect crop production. Developing ozone-tolerant rice (Oryza sativa L.) varieties is therefore essential to prevent yield losses in the future. The aims of this study were to assess genotypic variation for ozone tolerance in rice, to identify quantitative trait loci (QTL) conferring tolerance, and to relate QTLs to physiological tolerance mechanisms. The response of 23 varieties to elevated ozone (120 nl l–1) was assessed based on leaf bronzing and dry weight loss. The traditional variety ‘Kasalath’ was highly tolerant, whereas the modern variety ‘Nipponbare’ showed significant dry weight reductions. Using the Nipponbare/Kasalath/Nipponbare mapping population, six QTLs associated with tolerance to elevated ozone were identified, of which three were subsequently confirmed in Nipponbare/Kasalath substitution lines (SLs). Two QTLs associated with leaf bronzing were located on chromosomes three and nine. Kasalath alleles on chromosome three increased bronzing, while alleles on chromosome nine reduced bronzing. SLs carrying these contrasting QTLs differed significantly in leaf ascorbic acid (AsA) content when exposed to ozone, suggesting AsA as a principal antioxidant counteracting ozone-induced oxidative damage. A further confirmed QTL related to dry weight was located on chromosome eight, where the Kasalath allele increased relative dry weight. A SL carrying this QTL exhibited a less reduced net photosynthetic rate under ozone exposure compared with its recurrent parent Nipponbare. Although the effect of these QTLs on crop yield has not yet been established, their identification could be an important first step in developing ozone-tolerant rice varieties.
Key words: Ascorbic acid, oxidative stress, ozone, photosynthesis, QTL, rice, stomatal conductance
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
|---|
Tropospheric ozone is an air pollutant generated by the the photo oxidation of precursor gases, such as NOx, CO, and volatile organic compounds. While the emission of these precursor gases and, consequently, the tropospheric ozone concentration has decreased in recent years in developed regions such as the USA or the European Union, a rapid increase was seen in developing and emerging countries, especially in Asia (Jonson et al., 2006; Forster et al., 2007). For example, tropospheric ozone concentrations exceeding 80 nl l–1 occur regularly, even in rural areas of India or China (Cheung and Wang, 2001; Beig et al., 2007).
Earlier studies of the effect of ozone on crops focused on species such as soybeans or wheat, which have high significance in North America and Europe, where rising tropospheric ozone levels were first recognized. Only a few studies were undertaken with rice, although it is the most important food crop in Asia, if not in the world. Sensitivity of rice to ozone was demonstrated in growth chamber studies, where ozone fumigation reduced biomass production and grain yield due to decreasing light use efficiency (Kobayashi et al., 1995; Kobayashi and Okada, 1995). According to projections, ozone pollution may cause rice yield losses of up to 16% with no change in agricultural practices (Wang and Mauzerall, 2004; Ainsworth, 2008), which would put food security in Asia at substantial risk.
The uptake of ozone into the leaf mesophyll occurs mainly through the stomata during photosynthetic gas exchange (Fiscus et al., 2005). Adverse effects on plant photosynthesis were identified as a major factor limiting crop yields under high ozone levels. In wheat and beans, ozone exposure was shown to reduce the photosynthetic rate, mostly due to a loss of carboxylation efficiency (Farage et al., 1991; Morgan et al., 2003, 2004; Fiscus et al., 2005). A further mechanism by which ozone negatively affects plants is by oxidative damage. The breakdown of ozone in the apoplast is thought to lead to the formation of radical oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals. Such an ozone-induced oxidative burst results in tissue damage that produces visible leaf damage or ‘leaf bronzing’, possibly due to a ROS-induced cell death process (Baier et al., 2005; Fiscus et al., 2005; Rao and Davis, 2001).
Various mechanisms of tolerance to ozone have been suggested. Stomatal regulation is important in controlling gas influx into the leaf mesophyll and can help to exclude ozone from entering the leaf (Fiscus et al., 2005). Moreover, several ROS defence systems exist in plants, which can counteract oxidative damage caused by ozone. Among these defence systems are the ROS scavenging enzymes superoxide dismutase, catalase, and peroxidases, as well as a network of low molecular mass antioxidant compounds, such as ascorbate, gluthathione, phenolic compounds, and tocopherols (Blokhina et al., 2003). If genotypic variation for these tolerance mechanisms exists, it can be exploited for breeding tolerant crop varieties.
The use of tolerant genotypes is a powerful strategy in adapting rice production to rising ozone levels. Due to recent advances in the understanding of the rice genome, substantial progress has been made in the development of rice varieties with tolerance to various abiotic stress factors, including salinity, mineral deficiencies or toxicities, and flooding (Xu et al., 2006; Wissuwa et al., 2006; Ismail et al., 2007). By contrast, ozone stress has received far less attention, in spite of the threat of considerable yield losses described earlier. The aim of this study was therefore to (i) assess genotypic variation in the tolerance to ozone, (ii) to identify quantitative trait loci (QTLs) conferring tolerance and to confirm QTLs using chromosome segment substitution lines, and (iii) to test hypotheses regarding the physiological basis underlying QTL effects.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
|---|
Plant culture and ozone fumigation
In all experiments, seeds were germinated and placed on netted Styrofoam sheets floating on 0.5 mM CaCl2. One week after germination, seedlings were transferred to 75 l hydroponics tanks containing half-strength nutrient solution. After a further two weeks nutrient solutions were exchanged for full-strength solutions with the following element concentration: NH4NO3 1.42 mM, KH2PO4.2H2O 0.05 mM, K2SO4 0.5 mM, CaCl2.2H2O 1 mM, MgSO4.7H2O 1 mM, MnCl2.4H2O 9 µM, (NH4)6Mo7O24.4H2O 0.07 µM, H3BO3 18.5 µM, CuSO4.5H2O 0.16 µM, FeEDTA 36 µM, ZnSO4.7H2O 0.15 µM. The pH of the nutrient solutions was adjusted to 5.5 every other day. Ozone fumigation was initiated 2–3 d after the transfer of seedlings to full-strength nutrient solution. Hydroponic tanks with plants were placed in a 1.5x2.5 m growth chamber in the first experiment. The following experiments were carried out in one 1.5x3 m open top chamber placed in greenhouse cells. Plants were exposed to a target ozone concentration of 120 nl l–1 from 09.00 h to 16.00 h every day. Ozone was generated using an OES 10A ozone generator coupled with a Model 1150 ozone monitor (Dylec Inc., Osaka, Japan). Ozone was uniformly distributed inside the chamber using an air blower that forced ozone-enriched air through four perforated plastic tubes (3 cm diameter) that ran in parallel above the rice canopy at a distance of about 30–40 cm. In addition to the Model 1150 ozone monitor that regulated the OES 10A ozone generator to provide constant ozone levels, ozone concentrations were recorded by an independent ozone monitor (Series 500, Aeroqual Limited, Auckland, New Zealand) placed within the canopy. Air convection inside the chamber was measured using a Model 6003 air velocity meter (Kanomax Inc. Osaka, Japan). In each experiment, ozone fumigation was continued for 12–17 d. Control plants were cultured equivalently without ozone fumigation. Ozone concentrations were monitored periodically in the control and did not exceed 20 nl l–1.
Experiments
(i) The initial experiment to assess genotypic variation in response to ozone was conducted with a set of 23 diverse genotypes of rice (Oryza sativa L.) that included parental lines of various QTL mapping populations. Plants were grown in a growth chamber with an 11 h dark period and night temperatures of 24 °C. A 150 min transition period between darkness and daytime conditions was employed (same for day–night transition): temperatures and light intensities gradually increased until a maximum light intensity of 300–350 µmol m–2 s–1 at 30 °C was reached and maintained for 8 h. Relative air humidity was controlled at 80%. Ozone was introduced directly into the growth chamber using the above method and plants were exposed to ozone for 15 d from 09.00 h to 16.00 h.
(ii) QTL mapping was carried out using the permanent mapping population of 98 BC1F5 Nipponbare/Kasalath//Nipponbare backcross inbreed lines (BILs; Lin et al., 1998). Seeds of the BIL were obtained from the National Institute of Agrobiological Sciences (NIAS), Tsukuba, Japan. Nipponbare is a modern Japonica cultivar from Japan while Kasalath is an Indica landrace from India. This experiment was carried out from July to August 2007 in two adjacent climate-controlled greenhouse chambers under natural light with night/daytime temperature set at 25/30 °C, respectively. Plants were exposed to ozone for 12 d using the open top chamber as described above and average ozone concentration recorded with the external ozone monitor was 111.7 nl l–1.
(iii) The aim of this experiment was to confirm the effect of QTLs in Nipponbare/Kasalath chromosome segment substitution lines (SLs) that contained Kasalath chromosomal inserts at putative QTL positions in the genetic background of Nipponbare. (http://www.rgrc.dna.affrc.go.jp/ineNKCSSL54.html) Seeds were obtained from the National Institute of Agrobiological Sciences (NIAS), Tsukuba, Japan. The trial was carried out in a greenhouse from September to October 2007 with natural light conditions, a minimum temperature of 20 °C and the ventilation temperature set at 30 °C. Plants were exposed to ozone for 12 d and average ozone concentration was 124.4 nl l–1.
(iv) The objective of this experiment was to characterize the physiological basis of QTLs through experimental studies with Nipponbare and SLs that carried Kasalath alleles at QTL positions in the genetic background of Nipponbare. This trial was carried out in March in two adjacent climate-controlled greenhouse chambers under natural light with night/daytime temperatures set at 25/30 °C, respectively. Plants were exposed to ozone for 17 d and average ozone concentration was 120.2 nl l–1.
Phenotypic characterization
Visible ozone damage was quantified by assigning a leaf bronzing score (LBS) to the four topmost fully expanded leaves of each of four replicate plants. LBS ranged from zero (no visible damage) to nine (dead leaf) according to the scale used by Wissuwa et al. (2006). The number of tillers and plant height were measured after termination of the trial and dry weight was determined after drying for 48 h at 70 °C.
Mapping and confirmation of QTLs
For QTL mapping genotype data of 245 RFLP markers with an average distance of 4.8 cM were used. Genotype data are available online at http://rgp.dna.affrc.go.jp/E/publicdata/genotypedataBILs/genotypedata.html. The software package PLABQTL (Utz and Melchinger, 1996) was used for the detection of QTLs. This program employs composite interval mapping with cofactors using multiple regression. In a first step, simple interval mapping was performed and cofactors selected. F-to-enter value, i.e. the threshold for preselection of markers, was set at values ranging from six to ten to avoid selecting multiple markers linked to one QTL as cofactors. A log of the odds score (LOD) of 2.5 was used as the threshold for the declaration of QTLs. The effects of QTLs were confirmed using eight chromosome segment substitution lines (SLs), i.e. SL1, SL15, SL24, SL25, SL33, SL37, SL40, and SL41. Each of these lines contained chromosome inserts of Kasalath in the genetic background of Nipponbare at one or more putative QTLs (see Supplementary Fig. 2 at JXB online). Genotype data are available online at http://www.rgrc.dna.affrc.go.jp/data/NK-SL54-20030430.pdf.
Analysis of ascorbic acid (AsA)
Leaves were sampled twice after 4 d and 16 d of ozone exposure. The youngest fully expanded leaf was taken from each plant, immediately frozen in liquid nitrogen, and stored at –80 °C until analysis. Total AsA (including dehydroascorbic acid) was determined according to Badrakhan et al. (2004) with some modifications. Leaves were crushed in liquid nitrogen and 1.5 ml of 6% m-phosphoric acid containing 1 mM EDTA was added to around 30 mg of fresh leaf material. Samples were then put in an ultrasonic water bath for 20 min, centrifuged for 5 min at 12 000 rpm, and filtered through Millex-GV filters (Millipore Corporation, Bedford, MA). After adjusting pH to between 5 and 6, 10 µl of enzyme solution containing 0.77 units of ascorbate oxidase (EC 1.10.3.3, Sigma-Aldrich, St Louis, MO) in KH2PO4/K2HPO4 buffer (pH 6) was added and samples incubated for 5 min at room temperature. Subsequently, 30 µl of 15.4 mM deferrioxamin mesylate, 200 µl of methanol, and 400 µl of phosphate citrate buffer (pH 7.75) were added to each sample. As plant extracts contained pigments interfering with the spectrophotometer analysis, a background reference was prepared for each sample containing distilled water instead of methanol. After 30 min of incubation at room temperature absorbance was measured at 346 nm against a reagent blank using a Smartspec Plus spectrometer (Biorad, Hercules, CA). The absorption of the background reference was deducted from each sample absorption value.
Gas exchange measurements
Leaf gas exchange in experiment (iv) was measured using an open gas exchange system (LI-6400, Li-Cor, Lincoln, NE) with an integrated modulated fluorescence chamber head (LI-6400-40, Li-Cor). Measurements were made between 9.30 am and noon when photosynthesis was at its maximum. Ozone-treated plants were continuously exposed to 120 nl l–1 ozone during the measurements. Measurements were taken on completely sunny days only. Incident PAR at canopy height was determined before each measurement using an external quantum sensor attached to the leaf chamber (Li-190SA, Li-Cor), and the actinic light of the fluorescence chamber head was adjusted to the respective value (600–800 µmol m–2 s–1). Block temperature of the measuring chamber was set at 30 °C, corresponding to the air temperature during the measurements. On each measuring day, leaves were marked to enable subsequent measurements from the same leaf. As young rice leaves did not fully fill out the leaf chamber, leaf width was determined along with each measurement to correct values for stomatal conductance to H2O (gs) and photosynthetic rate (A) for effective leaf area. The flow rate was 500 µmol s–1 and inflowing air humidity (50–60%) was not conditioned. CO2 concentration was controlled to result in a reference CO2 concentration of 400 µl l–1 using a Li-Cor 6400-01 CO2 mixer. Measurements of stomatal conductance in experiments (ii) and (iii) were made using a SC-1 leaf porometer (Decagon Devices Inc., Pullman, WA).
Statistical analysis of data
The main purpose of these experiments was to detect genotypic differences under ozone exposure. Experiments were treated as split plots with ozone levels as the main plots and genotypes as the subplots. Within ozone treatments, experiments were arranged in a randomized complete block design with four replications. Analyses of variance were conducted by PROC MIXED in SAS, version 9.1, using Tukey's HSD to separate least-squares means.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
|---|
To test genotypic differences in response to ozone exposure, it was important to establish uniform conditions in the fumigation system. An ozone concentration of 120 nl l–1 was set as the target value for the treatment, because these ozone concentrations have been measured in rice fields across Asia and are likely to occur more frequently in the future. Through adjustments in wind speed, satisfactory homogeneity was achieved, with ozone values at canopy height ranging from a minimum of 90 to a maximum of 140 nl l–1, but mostly being close to 120 nl l–1 (Fig. 1). Ozone concentration at canopy height was recorded continuously, indicating that there was no diurnal drift, the ozone level thus being constant from 09.00 h to 16.00 h (see Supplementary Fig. S1 at JXB online).
|
Genotypic evaluation
Initial tolerance screening was conducted using a total of 23 rice varieties, including the parents of several QTL mapping populations. First visible leaf bronzing caused by ozone were observed 4–5 d after ozone fumigation had started. After 15 d, genotypes differed significantly in LBS, ranging from 0 to 5 (Fig. 2). Most genotypes showed slight to intermediate symptoms in the range of LBS 0.5 to 3. Only two genotypes, i.e. the traditional Indica variety Kasalath and the experimental line ‘NIL-Pup1’ (tolerant to phosphorus deficiency), did not produce visible leaf symptoms at all, while genotypes IR74 and Azucena showed excessive symptoms. Significant genotypic differences were also detected for relative dry weight (weight of ozone-treated plants as a proportion of control plants) with genotypes Nipponbare and Lemont being most sensitive to ozone (Fig. 1). Relative dry weight did not show a strong association with leaf bronzing score.
|
One purpose of this genotypic screen was to identify a suitable QTL mapping population. Kasalath ranked consistently as the most tolerant genotype for both LBS and relative dry weight, whereas Nipponbare was intermediate in terms of LBS but exhibited the most severe loss in dry weight under ozone exposure. The pronounced differences in tolerance indicators between these parental lines, and the conspicuously high tolerance ranking of Kasalath, led us to conclude that the Nipponbare/Kasalath mapping population would be suitable for further studies of genetic factors associated with ozone tolerance.
QTL mapping and confirmation
Mapping of QTLs associated with ozone tolerance was based on three phenotypic traits: leaf bronzing score (LBS), relative shoot dry matter, and stomatal conductance. In all three traits, the population of 98 backcross inbred lines exhibited much larger phenotypic variability than reflected in its parents Nipponbare and Kasalath (Fig. 3). While relative shoot dry matter and stomatal conductance showed more or less normal distribution, the frequency distribution of LBS was skewed towards lower values.
|
Using composite interval mapping four putative QTLs associated with LBS were detected, together explaining 52% of the phenotypic variation observed (Table 1). The QTLs were located near marker R1927 on chromosome three (OzT3), marker C1016 on chromosome four (OzT4), marker R521 on chromosome five (OzT5) and marker C1454 on chromosome nine (OzT9). Paradoxically, the tolerant parent Kasalath contributed only one tolerance allele (OzT9); at the remaining three QTLs Kasalath alleles increased LBS. For relative dry weight only one putative QTL (OzT8) was detected, located on chromosome eight near marker R2676. OzT8 explained 17.7% of the observed variation and the Kasalath allele had a positive effect on relative dry weight. No significant peak at marker R2676 was detected for dry weight in the control (data not shown), confirming that OzT8 was indeed a specific ozone tolerance QTL. A single QTL for stomatal conductance during ozone exposure (OzSC5) was detected on chromosome five near marker C1268. The QTL explained 14% of the phenotypic variation and the Kasalath allele increased conductance values.
|
To confirm the effect of putative QTLs, eight substitution lines (SL) containing introgressed chromosomal segments from Kasalath at putative QTL locations in the genetic background of Nipponbare (Supplementary Fig. S2 at JXB online) were used. SL15 carried the introgressed OzT3, SL1 contained the putative QTL OzT4, and SL33 harboured the Kasalath alleles at both OzT3 and OzT4. Two lines SL24 and SL25 had introgressions at OzT5, and SL24 additionally carried the Kasalath allele at OzSC5. The tolerance allele at the putative QTL OzT9 was present in SL40 and SL41. SL37 had inserts on chromosome eight and was used to confirm the dry weight related putative QTL OzT8.
All phenotypic traits were significantly influenced by ozone treatment, genotype, and treatment by genotype interactions (Table 2). In addition to producing leaf bronzing, the ozone treatment reduced tiller number and root and shoot dry weight. Kasalath was the most tolerant genotype as it was in both previous experiments, with Nipponbare being intermediate (Fig. 4). The effect of putative QTLs was tested by comparing least-square means of each SL to the recurrent parent Nipponbare (Fig. 4). A significant reduction in LBS compared to Nipponbare was observed in SL40 (P < 0.01) and SL41 (P < 0.05), confirming the positive effect of Kasalath alleles at OzT9. SL15 had a significantly higher LBS compared to Nipponbare (P < 0.01) thus confirming the negative effect of the Kasalath alleles at locus OzT3. Inconclusive results were obtained for OzT4 since SL1 was not significantly different from Nipponbare, while SL33 carrying both OzT3 and OzT4, showed significantly stronger symptoms (P < 0.05). However, it is likely that this effect was caused by the presence of OzT3. Contradictory results were also obtained regarding the effect of OzT5, as no difference was seen between SL24 and the recurrent parent, while SL25 had reduced LBS which contradicted estimated allelic effect in the QTL mapping (Table 1). It can thus be concluded that two out of four putative QTLs for leaf bronzing could be confirmed using SLs: OzT3, with Kasalath alleles increasing LBS, and OzT9 with Kasalath alleles reducing LBS. An additional QTL that could be confirmed was OzT8. SL37 had Kasalath inserts at this locus and its higher relative dry weight compared to Nipponbare (P < 0.05) suggested that Kasalath alleles at OzT8 prevented dry weight loss due to ozone exposure. None of the SLs differed significantly from Nipponbare in stomatal conductance. Thus the effect of QTL OzSC5 could not be confirmed.
|
|
Physiological basis of tolerance QTLs
Having identified and confirmed QTLs, a subsequent experiment using SLs and their recurrent parent Nipponbare was aimed at identifying the physiological basis of QTL effects. SLs carrying Kasalath alleles at QTL positions in the genetic background of Nipponbare were compared to their recurrent parent Nipponbare (see Supplementary Fig. 2 at JXB online). This approach allowed us to attribute phenotypic differences to the isolated effect of QTLs. Our hypotheses were that (i) QTLs OzT3 and OzT9 (LBS) are associated with leaf ascorbic acid (AsA) status and that (ii) OzT8 (dry weight) is associated with stomatal conductance and the photosynthetic rate. SL15 and SL41, carrying introgressions at OzT3 and OzT9 respectively, were used to investigate the effect of these two QTLs on leaf AsA status. Leaf samples taken after 4 d of ozone exposure exhibited significant genotypic differences in AsA content (Fig. 5). While AsA was deficient in intolerant SL15, the tolerant SL41 had significantly elevated AsA content compared to Nipponbare. Control plants had higher AsA level than ozone-treated plants and did not exhibit significant genotypic differences. A slightly different picture was obtained after 16 d of ozone exposure (Fig. 5): SL41 and Nipponbare had similar AsA content in the ozone treatment, while the intolerant SL15 was AsA deficient. The fact that SL41 did not show enhanced AsA content compared to Nipponbare at this point concurred with the gradual development of leaf symptoms in this genotype after 12 d of ozone exposure (data not shown). Similar to day 4, AsA level was generally lower in ozone-treated leaves than in control plants.
|
To investigate whether dry weight loss under ozone exposure was more closely related to reduced stomatal conductance or photosynthetic rate, Nipponbare was compared to SL37 containing QTL OzT8 for high relative dry weight. Both net photosynthetic rate (A) and stomatal conductance to water vapour (gs) dropped significantly when plants were exposed to ozone (Fig. 6; Table 3). The association between dry weight and A was stronger than that between dry weight and gs (Fig. 6). All three variables, i.e. dry weight, A, and gs, showed significant treatment by variety interaction, being less reduced in SL37 than in Nipponbare under ozone exposure (Table 3). The highest significance in treatment by variety interaction was seen in A, suggesting that it was closely linked to the presence of QTL OzT8.
|
|
Leaves of control plants also showed some loss in A that was possibly due to ageing (see drop in YLd2 at day 14; Fig. 7), but genotypes showed a similar pattern in the absence of ozone. Under ozone exposure even young leaves had reduced A but, in addition, it appeared that the ageing process was accelerated, particularly in Nipponbare. After 14 d of ozone exposure, only a small difference between genotypes was observed in the youngest and the oldest leaves, whereas a significant difference was seen in the middle leaves (Fig. 7). This suggests that differences between genotypes occurred mostly in a period of transition from a healthy leaf to a severely damaged leaf.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
|---|
Ozone concentrations around 120 nl l–1 have already been measured in rice-producing peri-urban and rural sites in China and India (Cheung and Wang, 2001; Beig et al., 2007; Wang et al., 2007). Therefore, our experiments were conducted at this realistic ozone concentration. This is in contrast to earlier attempts of tolerance screening and physiological studies in rice that were carried out at much higher concentrations of 300–400 nl l–1 to induce acute stress (Lin et al., 2001; Sohn et al., 2002). In spite of the relatively low concentrations used, the damage observed in terms of leaf bronzing and loss of biomass and photosynthetic capacity, was substantial. Pronounced genotypic differences in ozone tolerance suggest that there is scope for breeding tolerant rice varieties.
A leaf bronzing score (LBS) has been used frequently as a phenotypic parameter to evaluate genotypic differences in rice for tolerance to various abiotic stresses such as zinc (Zn) deficiency, salinity or iron toxicity (Quijano-Guerta et al., 2002; Sohn et al., 2002; Takehisa et al., 2006). Two QTL (OzT3 and OzT9) for LBS under ozone exposure were mapped and confirmed. For OzT3 the allele increasing LBS was derived from the tolerant parent which seems paradoxical, but similar results were reported for LBS caused by Zn deficiency (Wissuwa et al., 2006) and salinity (Takehisa et al., 2006). The QTL associated with leaf bronzing under salinity had been mapped in the same mapping population at the exact same location on chromosome three as OzT3. This would suggest that leaf bronzing under different abiotic stresses could be due to similar physiological mechanisms that are under common genetic control. In fact, genotypes that exhibited high LBS under ozone stress are also sensitive to other types of abiotic stress. In particular, this applies to IR74 (Fig. 2), which shows considerable leaf bronzing under zinc deficiency (Wissuwa et al., 2006). Correspondingly, the Zn deficiency-tolerant recombinant inbred line RIL46, derived from a cross between IR74 and the traditional Indica variety Jalmagna, was also tolerant to ozone (Fig. 2).
Leaf bronzing reflects the formation of radical oxygen species (ROS) that induce leaf damage. Various tolerance mechanisms could be responsible for the observed genotypic differences in leaf bronzing, probably related to the plant's ROS defence system. Antioxidant enzymes such as superoxide dismutases, catalase, and peroxidases, as well as low molecular weight antioxidants such as ascorbic acid (AsA), glutathione, tocopherols, and carotenoids have been linked to the ROS defence response (Blokhina et al., 2003; Apel and Hirt, 2004). Contrasting results have been reported as to how these ROS defence systems relate to ozone tolerance. Comparing a tolerant and an intolerant rice variety, Lin et al. (2001) showed that H2O2 levels and ROS scavenging enzyme activities were higher in an ozone-sensitive genotype. This suggests that the differences in ROS stress level formed in the leaves rather than the activity of ROS scavenging enzymes accounted for tolerance. On the other hand, Conklin and Barth (2004) reported that AsA, and especially the apoplastic AsA level, showed significant positive correlation with ozone tolerance in a wide range of plant species, a result that could confirm our analyses of leaf AsA content in SL15, SL41 and Nipponbare (Fig. 5). In particular the low level observed in intolerant SL15 indicates that a lack of AsA would lead to the formation of leaf symptoms. However, significant genotypic differences did not occur under non-stress conditions, where the AsA level was generally higher than in ozone-treated plants. This suggests that the AsA pool is gradually depleted when plants encounter stress, and that the ability to restore the AsA pool accounts for genotypic differences in leaf bronzing in the selected rice genotypes.
Leaf bronzing is a generally accepted criterion for assessing tolerance to several abiotic stresses, however, previous studies on zinc deficiency indicated that LBS was only weakly related to biomass production and crop yield (Wissuwa et al., 2006). A more direct relationship to yield might be expected from the additional parameters relative dry weight and stomatal conductance, for which the meta-analysis conducted by Ainsworth (2008) typically found a reduction in experiments that detected yield losses in rice under ozone exposure. Reduction in biomass in the present study was similar to earlier reports on rice and other crops (Fiscus et al., 2005; Morgan et al., 2006; Ainsworth, 2008). Because stomatal limitations are thought to affect photosynthesis and thus plant growth under ozone exposure (Ashmore, 2005), initially, a close association between dry weight and stomatal conductance was expected, and, possibly, overlapping tolerance QTLs for both traits. However, QTL analysis of stomatal conductance remained inconclusive. In part, this may be caused by methodological constraints, as distortions caused by diurnal variations are inevitable if stomatal conductance is measured in a large number of plants. But apart from these methodological considerations, stomatal conductance may not be the major limitation to photosynthesis despite being strongly affected by ozone. Genotypic differences in dry weight under ozone exposure between SL37 and Nipponbare were resolved clearly when plotted against net photosynthetic rate, but weaker association was found for stomatal conductance (Fig. 6). Probably factors affecting carboxylation efficiency accounted for genotypic differences in photosynthetic rate and ultimately biomass accumulation. Reduced quantity of active Rubisco was previously reported to be the main cause of decreased photosynthetic rate under ozone stress in wheat, while stomatal limitations were insignificant (Farage et al., 1991; Farage and Long, 1999). That the effect observed in SL37 might be associated with carboxylation is further corroborated by the overlap of a QTL for Rubisco content in rice (Ishimaru et al., 2001) with our QTL OzT8.
Loss of photosynthetic capacity was particularly pronounced in older rice leaves exposed to ozone for an extended period of time, similar to observations reported by Morgan et al. (2004) in soybeans. But, even in control plants, older leaves showed a reduced photosynthetic rate which is a natural consequence of leaf ageing (Murchie et al., 2002). It is hypothesized that the reduction in photosynthetic capacity in ozone-stressed plants is due to a combination of accelerated leaf ageing and damage of the photosynthetic apparatus, and that these processes proceed more rapidly in intolerant Nipponbare compared with tolerant SL37.
|
Conclusion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
|---|
Rice seedlings exhibited substantial stress symptoms under ozone exposure that included visible leaf symptoms and reduced biomass and tillering. Significant natural genotypic variation for these traits was detected and may offer the possibility for breeding ozone-tolerant rice varieties. By QTL mapping, this genotypic variation was dissected into distinct loci that were independently confirmed using SLs. This QTL-based approach furthermore allowed us to test hypothesis regarding the physiological basis of QTL effects using SLs. QTLs for leaf bronzing significantly influenced the leaf ascorbic acid status. A dry weight-related QTL was more closely related to the net photosynthetic rate than to stomatal conductance. While these QTLs and the associated tolerance mechanisms were conclusive in this greenhouse study, their potential for increasing rice yields in ozone-affected areas needs to be assessed in further studies.
|
Supplementary data |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
|---|
Supplementary data can be found at JXB online.
Supplementary Fig. S1. Within canopy ozone concentrations as recorded continuously over a 7 d period using an independent ozone monitor (Series 500, Aeroqual Limited, Auckland, New Zealand).
Supplementary Fig. S2. Graphical genotypes of chromosome segment substitution lines used for the confirmation of QTLs; white boxes indicate Nipponbare alleles, black boxes indicate Kaslath alleles, and grey boxes indicate a QTL position where SLs carry the Kaslath alleles.
|
Acknowledgements |
|---|
The authors wish to thank Dr M Yano, NIAS, Tsukuba, Japan, for providing plant material. This study was partially financed by the Japan Society for the Promotion of Science, JSPS (MF).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
|---|
Ainsworth EA. Rice production in a changing climate: a meta-analysis of responses to elevated carbon dioxide and elevated ozone concentration. Global Change Biology (2008) 14:1642–1650.[CrossRef][Web of Science]
Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology (2004) 55:373–399.[CrossRef][Medline]
Ashmore MR. Assessing the future global impacts of ozone on vegetation. Plant, Cell and Environment (2005) 28:949–964.[CrossRef]
Baier M, Kandlbinder A, Golldack D, Dietz KJ. Oxidative stress and ozone: perception, signalling and response. Plant, Cell and Environment (2005) 28:1012–1020.[CrossRef]
Badrakhan CD, Petrat F, Holzhauser M, Fuchs A, Lomonosova EE, de Groot H, Kirsch M. The methanol method for the quantification of ascorbic acid and dehydroascorbic acid in biological samples. Journal of Biochemical and Biophysical Methods (2004) 58:207–218.[CrossRef][Web of Science][Medline]
Beig G, Gunte S, Jadhav DB. Simultaneous measurements of ozone and its precursors on a diurnal scale at a semi urban site in India. Journal of Atmospheric Chemistry (2007) 57:239–253.[CrossRef][Web of Science]
Blokhina O, Virolainen E, Fagerstedt KV. Antioxidants, oxidative damage and oxygen deprivation stress: a review. Annals of Botany (2003) 91:179–194.
Cheung VTF, Wang T. Observational study of ozone pollution at a rural site in the Yangtze Delta of China. Atmospheric Environment (2001) 35:4947–4958.
Conklin PL, Barth C. Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant, Cell and Environment (2004) 27:959–970.[CrossRef]
Farage PK, Long SP. The effects of O3 fumigation during leaf development on photosynthesis of wheat and pea: an in vivo analysis. Photosynthesis Research (1999) 59:1–7.[CrossRef][Web of Science]
Farage PK, Long SP, Lechner EG, Baker NR. The sequence of change within the photosynthetic apparatus of wheat following short term exposure to ozone. Plant Physiology (1991) 95:529–535.
Fiscus EL, Booker FL, Burkey KO. Crop responses to ozone: uptake, modes of action, carbon assimilation and partitioning. Plant, Cell and Environment (2005) 28:997–1011.[CrossRef]
Forster P, Ramaswamy V, Artaxo P, et al. Changes in atmospheric constituents and in radiative forcing. In: Climate change: the physical science basis. Contribution of the working group I to the fourth assessment report of the International Panel on Climate Change—Solomon S, Qin D, Manning Z, Chen Z, Marquis M, Averyt KB, Tignor M, Millers HL, eds. (2007) Cambridge, UK: Cambridge University Press. 149–152.
Ishimaru K, Kobayashi N, Ono K, Yano M, Ohsugi R. Are contents of Rubisco, soluble protein and nitrogen in flag leaves of rice controlled by the same genetics? Journal of Experimental Botany (2001) 52:1827–1833.
Ismail AM, Heurer S, Thomson MJ, Wissuwa M. Genetic and genomic approaches to develop germplasm for problem soils. Plant Molecular Biology (2007) 65:547–570.[CrossRef][Web of Science][Medline]
Jonson JE, Simpson D, Fagerli H, Solberg S. Can we explain trends in European ozone levels? Atmospheric Chemistry and Physics (2006) 6:51–66.[Web of Science]
Kobayashi K, Okada M. Effects of ozone on the light use of rice (Oryza sativa L.) plants. Agriculture Ecosystems and Environment (1995) 53:1–12.[CrossRef]
Kobayashi K, Okada M, Nouchi I. Effects of ozone on dry matter partitioning and yield of Japanese cultivars of rice (Oryza sativa L.). Agriculture Ecosystems and Environment (1995) 53:109–122.[CrossRef]
Lin D-I, Lur H-S, Chu C. Effects of abscisic acid on ozone tolerance of rice (Oryza sativa L.) seedlings. Plant Growth Regulation (2001) 35:295–300.[CrossRef][Web of Science]
Lin SY, Sasaki T, Yano M. Mapping quantitative trait loci controlling seed dormancy and heading date using backcross inbred lines in rice, Oryza sativa L. Theoretical and Applied Genetics (1998) 96:997–1003.[CrossRef][Web of Science]
Morgan PB, Ainsworth EA, Long SP. How does elevated ozone impact soybean? A meta-analysis of photosynthesis, growth and yield. Plant, Cell and Environment (2003) 26:1317–1328.[CrossRef]
Morgan PB, Bernacchi CJ, Ort DR, Long SP. An in vivo analysis of the effect of season-long open-air elevation of ozone to anticipated 2050 levels on photosynthesis in soybean. Plant Physiology (2004) 135:2348–2357.
Morgan PB, Mies TA, Bollero GA, Nelson RL, Long SP. Season-long elevation of ozone concentration to projected 2050 levels under fully open-air conditions substantially decreases the growth and production of soybean. New Phytologist (2006) 170:333–343.[CrossRef][Web of Science][Medline]
Murchie EH, Hubbart S, Chen Y, Peng S, Horton P. Acclimation of rice photosynthesis to irradiance under field conditions. Plant Physiology (2002) 130:1999–2010.
Quijano-Guerta C, Kirk GJD, Portugal AM, Bartolome VI, McLaren GC. Tolerance of rice germplasm to zinc deficiency. Field Crops Research (2002) 76:123–130.[CrossRef][Web of Science]
Rao MV, Davis KR. The physiology of ozone induced cell death. Planta (2001) 213:682–690.[CrossRef][Web of Science][Medline]
Sohn JK, Lee JJ, Kwon YK, Kim KM. Varietal differences and inheritance of resistance to ozone stress in rice (Oryza sativa L.). SABRAO Journal of Breeding and Genetics (2002) 34:65–71.
Takehisa H, Ueda T, Fukuta Y, Obara M, Abe T, Yano M, Yamaya T, Kameya T, Higashitani A, Sato T. Epistatic interaction of QTLs controlling leaf bronzing in rice (Oryza sativa L.) grown in a saline paddy field. Breeding Science (2006) 56:287–293.[CrossRef][Web of Science]
Utz HF, Melchinger A. PLABQTL: a program for composite interval mapping of QTL. Journal of Quantitative Trait Loci (1996) 2. Paper1.
Wang X, Manning W, Feng Z, Zhu Y. Ground level ozone in China: distribution and effect on crop yields. Environmental Pollution (2007) 147:394–400.[CrossRef][Medline]
Wang X, Mauzerall DL. Characterizing distributions of surface ozone and its impact on grain production in China, Japan and South Korea: 1990 and 2020. Atmospheric Environment (2004) 38:4383–4402.[CrossRef]
Wissuwa M, Ismail A, Yanagihara S. Effects of zinc deficiency on rice growth and genetic factors contributing to tolerance. Plant Physiology (2006) 142:731–741.
Xu K, Xu X, Fukao T, Canlas P, Maghirang-Rodriguez R, Heurer S, Ismail AM, Bailey-Serres J, Mackill DJ. Sub1 is an ethylene response factor like gene that confers submergence tolerance in rice. Nature (2006) 442:705–708.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||