Effects of low temperature on photoinhibition and singlet oxygen production in four natural accessions of Arabidopsis

Abstract

Low temperature decreases PSII damage in vivo, confirming earlier in vitro results. Susceptibility to photoinhibition differs among Arabidopsis accessions and moderately decreases after 2-week cold-treatment. Flavonols may alleviate photoinhibition. The rate of light-induced inactivation of photosystem II (PSII) at 22 and 4 °C was measured from natural accessions of Arabidopsis thaliana (Rschew, Tenela, Columbia-0, Coimbra) grown under optimal conditions (21 °C), and at 4 °C from plants shifted to 4 °C for 2 weeks. Measurements were done in the absence and presence of lincomycin (to block repair). PSII activity was assayed with the chlorophyll a fluorescence parameter F_v/F_m and with light-saturated rate of oxygen evolution using a quinone acceptor. When grown at 21 °C, Rschew was the most tolerant to photoinhibition and Coimbra the least. Damage to PSII, judged from fitting the decrease in oxygen evolution or F_v/F_m to a first-order equation, proceeded more slowly or equally at 4 than at 22 °C. The 2-week cold-treatment decreased photoinhibition at 4 °C consistently in Columbia-0 and Coimbra, whereas in Rschew and Tenela the results depended on the method used to assay photoinhibition. The rate of singlet oxygen production by isolated thylakoid membranes, measured with histidine, stayed the same or slightly decreased with decreasing temperature. On the other hand, measurements of singlet oxygen from leaves with Singlet Oxygen Sensor Green suggest that in vivo more singlet oxygen is produced at 4 °C. Under high light, the PSII electron acceptor Q_A was more reduced at 4 than at 22 °C. Singlet oxygen production, in vitro or in vivo, did not decrease due to the cold-treatment. Epidermal flavonols increased during the cold-treatment and, in Columbia-0 and Coimbra, the amount correlated with photoinhibition tolerance.

Keywords: Acclimation; Charge recombination; Chilling stress; Cold-hardening; Photodamage; Photoinactivation; Reactive oxygen species; SOSG.

Fig. 1

Photoinhibition at 22 °C (a) or at 4 °C (b, c) in the absence of lincomycin, quantified by the chlorophyll a fluorescence parameter F_v/F_m. F_v/F_m was measured from detached leaves of four non-cold-treated (NT; a, b) or cold-treated (CT; c) A. thaliana accessions, at different time points during the 45-min illumination (PPFD 2000 µmol m⁻² s⁻¹), after subsequent 30-min dark incubation. The error bars show standard deviations (SD) from at least three biological replicates. Statistically significant differences at any time-point between the indicated accessions are marked with **P < 0.05 or ***P < 0.01. The control values of F_v/F_m (± SD) were 0.82 (0.03), 0.80 (0.03), 0.82 (0.03) and 0.76 (0.05) for Rschew (R), Tenela (T), Columbia-0 (Col) and Coimbra (Co), respectively, in a, 0.82 (0.03), 0.82 (0.03), 0.81 (0.04) and 0.79 (0.06) in b, and 0.82 (0.03), 0.79 (0.04), 0.77 (0.08) and 0.79 (0.06) in c

Fig. 2

Photoinhibition at 22 °C (a) or at 4 °C (b, c) in the presence of lincomycin, quantified by the chlorophyll a fluorescence parameter F_v/F_m. F_v/F_m was measured from detached leaves of four non-cold-treated (NT; a, b) or cold-treated (CT; c) A. thaliana accessions, at different time points during the 45-min illumination (PPFD 2000 µmol m⁻² s⁻¹), after subsequent 30-min dark incubation. The error bars show SD from at least three biological replicates. The lines show a fit to the first order reaction equation. The control values of F_v/F_m (± SD) were 0.82 (0.03), 0.79 (0.05), 0.83 (0.01) and 0.78 (0.04) for Rschew, Tenela, Columbia-0 and Coimbra, respectively, in a, 0.80 (0.01), 0.77 (0.02), 0.81 (0.03) and 0.79 (0.03) in b and 0.82 (0.03), 0.77 (0.07), 0.79 (0.06) and 0.79 (0.06) in c

Fig. 3

Photoinhibition at 22 °C (a) or at 4 °C (b, c) in the presence of lincomycin, quantified by the light-saturated oxygen evolution of PSII in the presence of an artificial electron acceptor. PSII activity was measured from thylakoid membranes isolated from leaves of four non-cold-treated (NT; a, b) or cold-treated (CT; c) A. thaliana accessions, at different time points during the 45-min illumination (PPFD 2000 µmol m⁻² s⁻¹). The error bars show SD from at least four biological replicates. The lines show a fit to the first order reaction equation. The control values in µmol O₂ (mg chlorophyll)⁻¹ h⁻¹ (± SD) were 160 (37.5), 162 (26.4), 180 (20.2) and 132 (31.2) for Rschew, Tenela, Columbia-0 and Coimbra, respectively, in a, 169 (67.6), 148 (67.1), 162 (37.4) and 135 (24.7) in b and 210 (21.7), 186 (22.9), 164 (28.7) and 162 (37) in c

Fig. 4

Chlorophyll (a) and flavonol (b) contents measured from intact leaves of A. thaliana accessions (Rschew, Tenela, Columbia-0 and Coimbra) after 7 or 14 days, as indicated, of cold-treatment at 4 °C (CT) or from control plants of similar ages grown at 21 °C (NT). The error bars show SD values from at least four biological replicates. Statistically significant differences are marked with **P < 0.05 or ***P < 0.01 on top of the horizontal lines that show between which samples the difference is significant; the horizontal curly bracket indicates a whole group of four accessions. The significance of the differences between different accessions are shown only within the same treatment group, and significances between NT and CT plants are shown only between the corresponding age groups

Fig. 5

Net CO₂ assimilation rates at 22 °C during 42-min illumination in high light (PPFD 2000 µmol m⁻² s⁻¹), measured from intact light-acclimated leaves of non-cold-treated (a) or cold-treated (b) A. thaliana accessions (Rschew, Tenela, Columbia-0 and Coimbra). The plants were illuminated with a light intensity close to that of growth light (PPFD 100 µmol m⁻² s⁻¹) for 1 min after which the high light was switched on (at the time point 0). The lines show averages and the colored areas show SD values from at least three biological replicates

Fig. 6

Production of ¹O₂ by isolated thylakoid membranes in vitro (a), by a methylene blue solution (b) and by leaf disks in vivo (c, d). The measurements were done at 20–22 °C or 4 °C, as indicated, from A. thaliana accessions (Rschew, Tenela, Columbia-0 and Coimbra) grown at 21 °C (NT), cold-treated for 2 weeks (CT), grown and developed at 4 °C (CD) or grown in high light at 20 °C (HL). Production of ¹O₂ in vitro (a) was measured with a histidine-based method in high light (PPFD 4000 µmol m⁻² s⁻¹). ¹O₂ production by methylene blue and by A. thaliana leaves in vivo (b–d) was measured as an increase in SOSG fluorescence after 0–45 min illumination with red light of PPFD 2000 µmol m⁻² s⁻¹ (b, c) or 1000 µmol m⁻² s⁻¹ (d). After each illumination period, SOSG fluorescence was excited with 500 nm light and recorded at 535–555 nm. The error bars show SD values from at least three biological replicates. Statistically significant differences in a are marked with **P < 0.05. The significances of the differences between different accessions are shown only within the same treatment group, and significances between NT and CT plants are shown only between the corresponding temperatures

Fig. 7

Normalized thermoluminescence bands recorded in the absence (a, c) or in the presence of DCMU (b, d) from thylakoid membranes of non-cold-treated (NT; a, b) or cold-treated (CT; c, d) A. thaliana accessions (Rschew, Tenela, Columbia-0 and Coimbra). The colored areas show SD values from at least three biological replicates. Maximum luminescence intensities (arbitrary units; ± SD) were 2.1 (0.60), 1.8 (0.45), 1.7 (0.15) and 2.8 (0.21) for Rschew, Tenela, Columbia-0 and Coimbra, respectively, in a, 2.5 (0.77), 2.2 (0.23), 2.2 (0.17) and 2.6 (0.39) in b, 2.0 (0.26), 1.5 (0.20), 1.4 (0.25) and 1.5 (0.2) in c, and 1.8 (0.65), 1.2 (0.41), 1.6 (0.16) and 1.9 (0.08) in d

Fig. 8

Chlorophyll a fluorescence parameters (a–f) and oxygen evolution (f) during 45-min illumination at 22 °C (a, d, f) or at 4 °C (b, c, e, f) measured from detached leaves of non-cold-treated (NT; a, b), cold-treated for 2 weeks (CT; c) or grown in high light at 20 °C (HL; d, e) A. thaliana accession Columbia-0. PPFD was 2000 µmol m⁻² s⁻¹ in a–c and 1000 µmol m⁻² s⁻¹ in d–f. F₀ and F_m were measured after 30 min dark-acclimation [time point 0 min (30 min D)] and all other parameters were measured in the light, 15, 30 or 45 min after switching on the light, as indicated (a–e). F is the fluorescence yield under illumination. F_v/F_m is the maximum quantum yield of PSII electron transfer, calculated as (F_m − F₀)/F_m. ɸ_II is the quantum yield of PSII electron transfer in the light, calculated as (F_m' − F)/F_m'. F₀′ is the value of F₀ in the light, calculated as 1/(1/F₀ − 1/F_m + 1/F_m'). qP and qL are estimates of photochemical quenching; qP is calculated as (F_m' − F)/(F_m' − F₀′) and qL as qP × F₀′/F. F₀, F_m and F_m' values may differ between treatment groups due to different settings of the fluorometer. Occasional below-zero qP or qL values in (a–c) were interpreted as zeros. HL Columbia-0 leaves were incubated overnight with the petioles in lincomycin (d–f), and photoinhibition after the 45-min illumination was quantified by the chlorophyll a fluorescence parameter F_v/F_m from leaves after 30 min in dark and by the light-saturated oxygen evolution of PSII in the presence of an artificial electron acceptor measured from thylakoids isolated from the illuminated leaves (f). The error bars show SD from at least three biological replicates. In d, e F₀′, qP and qL have been calculated from averages

Fig. 9

Rate constants of photoinhibition (quantified as a loss of oxygen evolution capacity of PSII) plotted against the amount of epidermal flavonols of four A. thaliana accessions (Rschew, Tenela, Columbia-0 and Coimbra). Values are measured after 2 weeks of cold-treatment at 4 °C (CT) or from control plants of similar ages grown at 21 °C (NT). The data are from Fig. 4 and from Table 1