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First published online June 1, 2004; 10.1104/pp.104.041293 Plant Physiology 135:1040-1049 (2004) © 2004 American Society of Plant Biologists Salicylate Accumulation Inhibits Growth at Chilling Temperature in Arabidopsis1Institute of Biological Sciences, University of Wales, Aberystwyth, SY23 3DA, United Kingdom
The growth of Arabidopsis plants in chilling conditions could be related to their levels of salicylic acid (SA). Plants with the SA hydroxylase NahG transgene grew at similar rates to Col-0 wild types at 23°C, and growth of both genotypes was slowed by transfer to 5°C. However, at 5°C, NahG plants displayed relative growth rates about one-third greater than Col-0, so that by 2 months NahG plants were typically 2.7-fold larger. This resulted primarily from greater cell expansion in NahG rosette leaves. Specific leaf areas and leaf area ratios remained similar in both genotypes. Net assimilation rates were similar in both genotypes at 23°C, but higher in NahG at 5°C. Chlorophyll fluorescence measurements revealed no PSII photodamage in chilled leaves of either genotype. Col-0 shoots at 5°C accumulated SA, particularly in glucosylated form. SA in NahG shoots showed similar tendencies at 5°C, but at greatly depleted levels. Catechol was not detected as a metabolite of the NahG transgene product. We also examined growth and SA levels in SA signaling and metabolism mutants at 5°C. The partially SA-insensitive npr1 mutant displayed growth intermediate between NahG and Col-0, while the SA-deficient eds5 mutant behaved like NahG. In contrast, the cpr1 mutant at 5°C accumulated very high levels of SA and its growth was much more inhibited than wild type. At both temperatures, cpr1 was the only SA-responsive genotype in which oxidative damage (measured as thiobarbituric acid-reactive substances) was significantly different from wild type.
Salicylic acid (SA) has received much attention due to its association with economically important plant responses to disease and other stresses. Detailed evidence implicates SA in PR gene expression, systemic acquired resistance, and the hypersensitive response (Kunkel and Brooks, 2002  ). SA also seems to be involved in responses to abiotic stresses, such as ozone (Sharma et al., 1996; Rao and Davis, 1999; Koch et al., 2000), salt and osmotic stress (Borsani et al., 2001; Molina et al., 2002; Shim et al., 2003), UV-B (Surplus et al., 1998), drought (Senaratna et al., 2000; Nemeth et al., 2002), paraquat (Kim et al., 2003), and heat (Dat et al., 1998a, 1998b, 2000; Lopez-Delgado et al., 1998a; Senaratna et al., 2000; Larkindale and Knight, 2002; Clarke et al., 2004). Stress-influenced developmental transitions, including flowering (Hatayama and Takeno, 2003; Martinez et al., 2004), tuberization (Lopez-Delgado and Scott, 1997), and senescence (Morris et al., 2000), may also involve SA.
Cold is one of the most important limitations to crop productivity and species distribution. Freezing (subzero) or chilling (low positive) temperatures can cause injury or reduced growth depending on the cold tolerance of the species (Schneider et al., 1995
We have recently subjected a range of SA-related Arabidopsis genotypes to different temperatures (Clarke et al., 2004
The SA-related genotypes we tested came from pathogen defense research, but common pathways and components occur in biotic and abiotic stresses (Chen et al., 2002
The npr1 mutant was characterized as a nonexpresser of SA-inducible PR genes with reduced pathogen resistance (Cao et al., 1994
One example of a signal pathway gene functioning upstream of SA is the enhanced disease susceptibility mutant eds5 (Rogers and Ausubel, 1997
The cpr1 mutant, characterized as a constitutive expresser of PR genes, also seems to act upstream of SA, but, in contrast to eds5, it has elevated levels of SA (Bowling et al., 1994 These genotypes therefore represent a range of putative points in the SA signal transduction network. We describe here how their development and SA levels at 5°C indicate that this hormone may be one contributory factor in the low-temperature inhibition of growth in Arabidopsis.
Growth of NahG Plants at Chilling Temperature When Arabidopsis seedlings of the SA-deficient NahG and wild-type Col-0 genotypes grown in long days (16 h) were transferred to 5°C rather than incubated at 23°C, growth rates were markedly lower, as expected (Fig. 1A). Among the 5°C plants, however, a size differential in favor of NahG started to be apparent by the third week of chilling and became increasingly pronounced so that the mean biomass of NahG plants was typically 2.7-fold greater than wild type after 2 months (Fig. 1A). Relative growth rates (RGRs) were estimated as the gradient of ln (total plant biomass) over periods of exponential growth (minimum r2 of linear regression = 0.988). For the 5°C-grown plants, RGR over 8 to 35 d was significantly greater (P < 0.05) by 31% for NahG, at 0.0944 d–1 compared with 0.0719 d–1 for Col-0. RGRs for the 23°C-grown plants (over 6–21 d) were approximately threefold greater, but not significantly different between the genotypes (NahG = 0.267 d–1; Col-0 = 0.250 d–1).
The phenotypic differences between NahG and Col-0 plants at 5°C, shown after 7 weeks in Figure 1B, were consistently observed in over 20 experiments. NahG plants were also significantly larger (P < 0.001) in the short-day (8 h) conditions that would be more common in the natural environment at 5°C, but growth was even slower (by >2.5-fold) than in long days (data not shown). The most striking feature of NahG plants grown at 5°C was the relatively large leaf size (Fig. 1B). Total leaf area of both genotypes showed a strong linear correlation with plant biomass (minimum r2 = 0.995). The area of all rosette leaves (plants were not flowering) on NahG plants was 2.8-fold greater than wild type after 2 months of cold treatment (Fig. 2A). The number of leaves also appeared greater for NahG plants at 5°C (Fig. 2B). The ratio of NahG:Col-0 leaf numbers was, however, constant with time (regression line gradient = –0.0001) with a mean ± SE of 1.31 ± 0.04 (n = 9). (For 23°C controls, the equivalent ratio was 0.96 ± 0.03.) This suggests the greater expansion of NahG leaves simply caused more leaves to reach the macroscopically countable length of 1 mm, without a changed rate of production of leaf primordia.
The size difference between NahG and wild-type leaves at 5°C could be accounted for by cell expansion. Mean areas of fully expanded NahG leaves from plants kept 76 d at 5°C were 2.8-fold greater than wild type (Fig. 2C), while mean cross-sectional areas of abaxial epidermal cells of these NahG leaves were likewise 2.8-fold greater than wild type (Fig. 2D). In consequence, no difference could be found in the numbers of abaxial epidermal cells per leaf of the two genotypes (Fig. 2E). Specific leaf areas (area-to-dry matter ratio) of mature individual leaves did not differ significantly: Col-0 = 234 ± 6 cm2 g–1; NahG = 228 ± 7 cm2 g–1 (n = 9) after 46 d at 5°C. Leaf area ratio (LAR) is the ratio of leaf area to plant biomass. On chilling of NahG and Col-0 plants, LARs declined rapidly to values approximately 60% of those at 23°C (Fig. 3). However, LARs appeared similar in NahG and Col-0 (Fig. 3), consistent with a lack of significant difference found in regressions of leaf area against plant biomass in the two genotypes (not shown). This indicates that there was no gross morphological difference between chilled NahG and Col-0 plants apart from the larger size of the former.
Since RGR is the product of LAR and net assimilation rate (NAR; Poorter and Remkes, 1990 ), the similar LARs in both genotypes suggest the greater RGR of NahG at 5°C was due to a greater NAR. Over the period used to estimate RGR, mean NAR was significantly higher (P < 0.05) in NahG than Col-0 at 5°C (Fig. 4A). NARs were higher in plants at 23°C but not significantly different between genotypes (Fig. 4A).
Maximal efficiencies of PSII photochemistry were measured as the ratio of variable-to-maximum chlorophyll fluorescence (Fv/Fm), to assess the possible occurrence of PSII photodamage in chilled leaves (Maxwell and Johnson, 2000 ). No significant differences were found in Fv/Fm values, either between NahG and Col-0 leaves or between plants grown at 23°C or 5°C (Fig. 4B). The effects of chilling on free and glucosylated SA levels in NahG and Col-0 shoots were investigated using an isotope dilution liquid chromatography-mass spectrometry (LC-MS) method. As the rates of growth at 23°C and 5°C were very different, comparisons were made both at equal ages (Fig. 5A) and at equal stages of development (Fig. 5B). By either criterion, chilling induced accumulation of free SA and, to higher levels, of glucosyl SA. In the equal-age experiment in Figure 5A, plants kept 12 d at 5°C or 23°C reached minimum growth stages of 1.06 or 5.10, respectively. In the Col-0 shoots at 5°C, free and glucosyl SA levels were, respectively, 4.1- and 6.3-fold higher than at 23°C. SA levels were depleted in NahG shoots, so that at 5°C free and glucosyl SA contents were 10% and 26% of wild type. Chilling-induced increases in the low levels of free and glucosyl SA were still observed in NahG, however, to 1.5- and 4.8-fold higher than at 23°C. In the equal-stage experiment in Figure 5B, plants were grown to stage 1.08, taking 20 d at 5°C or 5 d at 23°C. In this experiment, the chilling treatment was therefore 8 d longer than in the equal-age experiment, and SA accumulation was more pronounced. In Col-0 shoots, free and glucosyl SA levels were, respectively, 20- and 49-fold higher at 5°C than at 23°C. In NahG shoots at 5°C, free and glucosyl SA increased to only 2.3- and 1.5-fold higher than at 23°C, and were only 4.8% and 1.2% of wild type at 5°C. The substantial chilling-induced accumulation of SA in Col-0 shoots was not an early response. In the first week of chilling we were unable to detect significant increases in SA (data not shown).
As the NahG gene product converts SA to catechol, which may exert biological effects itself (Van Wees and Glazebrook, 2003 ), we monitored the catechol [M-H]– ion at m/z 109 in the same LC-MS analyses as for SA. We were not able to detect catechol above the detection limit of approximately 0.4 µg/g fresh weight in either genotype at either temperature.
To explore further the low-temperature role of SA implied by the transgenic NahG line, we compared genotypes with mutations affecting SA signaling and metabolism: npr1, eds5, and cpr1. Like NahG, each of these mutants showed significant (P < 0.01) divergence from wild-type growth over several weeks at 5°C (Fig. 6, A and B). The npr1 mutant (which shows impaired sensitivity to SA in pathogen responses) displayed intermediate 5°C growth that was significantly different (P < 0.01) from both NahG and Col-0, reaching 120% of wild-type shoot biomass after 52 d, compared to 165% for NahG. The eds5 mutant (which exhibits greatly reduced SA accumulation on infection) showed relatively substantial 5°C growth that was significantly greater (P < 0.05) than npr1, but not significantly different (P = 0.26) from NahG. (Three comparable experiments failed to show significantly less growth in eds5 than NahG.) The enhanced low-temperature growth of NahG, npr1, and eds5 occurred despite the plants at 23°C showing slightly less growth than Col-0 (Fig. 6B). A markedly different growth pattern was observed in the SA-accumulating cpr1 mutant. This mutant has an environmentally conditioned dwarf phenotype (Stokes and Richards, 2002
SA contents of the genotypes after 42 d at 5°C were consistent with growth inhibition by this hormone (Fig. 6C). SA levels in Col-0 shoots were comparable to those found after 20 d at 5°C (compare with Fig. 5B), suggesting maximal wild-type accumulation had occurred. Again, SA occurred in NahG at fractions of wild-type levels (9% free and 1.5% glucosyl SA), and SA levels in eds5 were even lower (3.6% free and 0.7% glucosyl SA). In contrast, excessive accumulation of SA occurred in cpr1, with 100 µg g–1 of glucosyl SA (2.8-fold more than wild type), and free SA even more elevated in relative terms at 5 µg g–1 (8.3-fold more than wild type). In npr1 shoots, free SA was strongly elevated at 7.3-fold more than wild type, though glucosyl SA levels were similar to Col-0. Thus, free SA in npr1 deviated from the inverse correlation between SA and shoot biomass: r2 was 0.903 for a regression of free SA against biomass in the other genotypes, but fell to 0.550 when npr1 was included. No significant differences were found between mean Fv/Fm values for Col-0, NahG, npr1, and eds5 at either 5°C or 23°C, all values being in the range 0.777 to 0.787 (n = 10). The cpr1 plants, whose phenotype was suggestive of stress, were too small for this measurement, so another parameter of cellular damage was investigated.
Oxidative damage measured as thiobarbituric acid-reactive substances (TBARS) is widely used as a stress indicator in biological systems, including cold-treated plants (Taulavuori et al., 2001
Wild-type Arabidopsis shoots under chilling conditions slowly accumulated salicylate as free and glucosyl SA. Genotype comparisons indicated a strong negative correlation between growth rate and the levels or perception of SA. As our studies involved endogenous SA, production of a growth inhibitor appears to be a genuine physiological response to chilling. The growth-inhibitory properties of salicylates have already been noted without a specific explanation for a physiological role. Acetyl-SA at 100 µM retarded stem growth in potato microplants (Lopez-Delgado and Scott, 1997 ), such that Lopez-Delgado et al. (1998b) proposed its use as an alternative medium supplement to mannitol for slow-growth storage of potato germplasm. Growth of tobacco (Nicotiana tabacum) seedlings on 100 µM SA likewise reduced shoot biomass and leaf epidermal cell size (Dat et al., 2000). We found endogenous SA accumulation in chilled Arabidopsis plants had comparable effects: RGRs were substantially higher than wild type in SA-deficient NahG plants, whose leaves grew much larger due to greater cell expansion.
There is much current interest in cellular mechanisms of cold sensing and their transduction into physiological responses (Murata and Los, 1997
Insights into SA signaling in growth at chilling temperature were provided by SA-related mutants from pathogen defense research. The eds5 mutants had the same chilling phenotype as the similarly SA-deficient NahG transgenics, which, together with our negative catechol analyses, suggests that the NahG chilling phenotype was not due to SA degradation products, as proposed by Van Wees and Glazebrook (2003)
Growth of npr1 at 5°C was significantly greater than wild type, though less than NahG or eds5. NPR1 is an SA-activated regulator of gene expression in SAR (Fan and Dong, 2002
Clarke et al. (2000)
The inverse correlation between SA and growth in chilled plants was extended with the cpr1 mutant, which had high free and glucosyl SA levels and whose development was severely restricted at 5°C. Growth of cpr1 was also restricted at 23°C, though to a less extreme extent. TBARS assays of cpr1 gave higher readings than wild type at both 5°C and 23°C, indicating a greater propensity for oxidative damage in this mutant. The phenotype of cpr1 appears to be exceptionally sensitive to environmental conditions. Stokes and Richards (2002)
We assessed cellular damage using Fv/Fm and/or TBARS measurements, and cpr1 was the only genotype in which a significant difference from wild type could be demonstrated. Although differences might be revealed by other stress criteria, such as cellular redox state (Dat et al., 2000
In cpr1, on the other hand, the increased oxidative damage alongside growth inhibition reaffirms the relationship between SA and cellular stress. The collective data on SA and oxidative stress have a complex pattern, consistent with the type of model proposed by Rao and Davis (1999)
Previous studies of SA in low-temperature physiology have examined tolerance of treatments causing injury in the species concerned and have tended to use short treatments with exogenous SA (Janda et al., 1999
The effects of SA accumulation only partly explain the chilling-induced slowdown of growth. Temperature downshift from 23°C to 5°C caused an immediate growth reduction in both Col-0 and NahG. Indeed, no differences between these genotypes were visible for at least the first 2 weeks of chilling, and significant SA accumulation was not detectable until the second week. Numerous physiological effects of chilling have been identified, including potential disruption of all major components of photosynthesis (Allen and Ort, 2001
The slow development of SA-correlated growth differentials was thus superimposed on the immediate growth reduction seen upon chilling. This could reflect a hormonal role in dampening the potential for rapid reversals in growth rate under fluctuating environmental conditions, as growth rates tend to correlate inversely with frost resistance during cold acclimation, deacclimation, and reacclimation episodes (Rapacz, 2002
The association of slow or compact plant growth patterns with adverse environments has long been recognized, but the relationships are complex (Poorter and Remkes, 1990
Plant Materials and Growth Conditions
Arabidopsis ecotype Columbia (Col-0) was used as wild type and the transgenic and mutant lines were in this background. The 35S-NahG line was donated by Scott Uknes (Cropsolution, Research Triangle Park, NC). The npr1 and cpr1 mutants were from Xinnian Dong (Duke University, Durham, NC), while eds5 was from Fred Ausubel (Harvard Medical School, Boston, MA). Seeds sown in Levington Universal Extra compost (Scotts, Ipswich, UK) were vernalized for 1 week at 4°C, and then germinated in a glasshouse heated to a minimum 23°C, with a 16-h daily light period supplemented, if necessary, by 400-W sodium lamps. Experimental populations were at a mean growth stage of 1.06 ± 0.01 (Boyes et al., 2001
Dynamic growth analysis of Col-0 and NahG at 5°C and 23°C (Figs. 1–4A) was started at a mean growth stage of 1.04. Four plants, of each genotype at each temperature, were harvested at intervals, and the number of leaves >1 mm counted on each. Total leaf area per plant (A) was measured on a Delta-T Devices Area Measurement System (Cambridge, UK). Finally, oven-dried biomass of whole plants, including roots (W), was measured. Growth curves are shown in Figures 1 and 2 fitted by least-squares regression to a logistic function, W = a / (1 + be–ct), where t is time, and a, b, and c are constants. RGR was estimated as the regression line gradient of ln W against t over a period of exponential growth spanning five harvests. LAR was determined for each harvest as A/W, while NAR was derived as RGR/LAR (Poorter and Remkes, 1990 Epidermal cell measurements were made on photomicrographs of replicas obtained by evaporating a viscous acetone solution of cellulose acetate in contact with abaxial surfaces of fully expanded rosette leaves. Cross-sectional areas of 10 cells on each of five leaves per genotype were measured using PC_Image Version 2.2 software (Foster Findlay, Aberdeen, UK). The number of abaxial epidermal cells was estimated by dividing the area of each leaf by the mean area of its cells. Single-harvest measurements were also made of oven-dried biomass of whole shoots without roots (e.g. Fig. 6B).
Photochemical efficiency of PSII was measured as the ratio of variable-to-maximal chlorophyll fluorescence (Fv/Fm) in fully expanded attached leaves, following 10-min dark adaptation with leaf clips at the growth temperature. Measurements were made at a 685-nm excitation of 1000 µmol photons m–2 s–1 for 2 s with an Opti-Sciences OS-30 Continuous Source Chlorophyll Fluorometer (Tyngsboro, MA).
Shoot tissues (up to 0.5 g) were ground in liquid N2 and extracted for at least 3 h at 5°C in 20 mL 80% methanol with addition of an internal standard of d6-SA (98 atom %; C/D/N Isotopes, Pointe-Claire, Quebec, Canada). Samples were passed through filter paper and the methanol removed by rotary evaporation at 25°C. After sample centrifugation (13,000 rpm, 3 min), half of each supernatant was incubated overnight at 37°C with an equal volume of buffer (0.2 M sodium acetate, pH 4.5) in the presence of 10 units of almond
TBARS were assayed by a modification of the method of Hideg et al. (2003)
Significance of differences between pairs of sample means or regression lines was estimated by two-tailed t tests. Excel was used for all statistical calculations.
We are extremely grateful to Tallulah Crow and Jim Heald for technical assistance, and to Xinnian Dong and Scott Uknes for materials donated. Valuable contributions from Mike Humphreys (Institute of Grassland and Environmental Research, UK), Steve Neill and John Hancock (University of the West of England, UK), and Rob Darby are also gratefully acknowledged. Received February 19, 2004; returned for revision March 9, 2004; accepted March 9, 2004.
1 This work was supported by the Biotechnology and Biological Sciences Research Council. 
2 Present address: Biochemistry Department, University of Otago, P.O. Box 56, Dunedin, New Zealand. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041293. * Corresponding author; e-mail ias{at}aber.ac.uk; fax 44–1970–622350.
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ABSTRACT
RESULTS
-glucosidase EC 3.2.1.21 (NBS Biologicals, Huntingdon, UK). Glucosidase-treated and untreated fractions were each partitioned at pH 2 against equal volumes of ethylacetate. The organic phases were back-washed against H2O and reduced to dryness by rotary evaporation at 25°C. Samples were chromatographed at 30°C on a Waters Nova-Pak C18 cartridge (3.9 x 50 mm), using a 10% to 95% gradient of methanol in 2 mM formic acid over 15 min, at a flow rate of 0.5 mL min–1, on a Waters Alliance 2690 liquid chromatograph (Elstree, UK). Ten percent of the eluate was introduced into a Waters Micromass LCT electrospray ionization LC-MS in negative ion mode with a sample cone voltage of 30 V, capillary voltage of 2.0 kV, and an extraction voltage of 5 V. SA was quantified by calibration of the molar ratio between the [M-H]– ions at m/z 137 (natural SA) and m/z 141 (internal standard). SA glucoside was analyzed as the extra SA liberated in the glucosidase-treated fractions. The possible presence of catechol was also monitored in the LC-MS analyses using its m/z 109 [M-H]– ion calibrated against the d6-SA internal standard. The electrospray ionization detection was 5.6 times less sensitive for catechol than for SA, giving a detection limit of approximately 0.4 µg g–1.