Article Figures & Data
Figures
- Figure 1.
Phenotype of pxa1 Plants and Impact on Photosynthesis after Extended Darkness.
(A) Visible phenotype of pxa1, kat2, and Col-0 wild-type plants after exposure to 36 h of darkness at 24°C.
Pictures were taken 24 h after retransferring plants into regular day/night conditions.
(B) Photosynthetic parameters Fv/Fm ratio and ΦPSII, indicating intactness of PSII measured in intact leaves of wild-type and pxa1-2 plants exposed to increasing periods of darkness. Average ± se (n = 10).
(C) Immunoblots of total leaf protein using specific antibodies against D1, D2, and LHCB2 protein. Coomassie shows protein stain of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Numbers on top refer to hours exposed to darkness.
- Figure 2.
Impact of Individually Covered Leaves on Occurrence of the pxa1 Dark Treatment Phenotype.
Col-0 and pxa1-2 plants grown at a regular 16/8-h (day/night) cycle, where individual leaves were darkened for 64 h before aluminum covers were removed (left panel) and leaves reexposed to the regular day/night cycle for 24 h. No necrotic lesions could be detected in any of the dark-treated leaves.
[See online article for color version of this figure.]
- Figure 3.
Effects of Prolonged Dark Treatment on Fatty Acid Metabolism of pxa1 Plants.
Lipids were extracted from leaves of 21-d-old plants pregrown in a 16/8-h (day/night) cycle and exposed to darkness for the time indicated. Plants were harvested at the end of the dark treatment without reexposure to light. The values present the mean of at least three biological replicates, and error bars indicate se. Square: Significantly different values between wild-type and pxa1-2 plants at the same incubation time (P ≤ 0.05). Triangle: Significantly different within one genotype between the incubation time indicated and time 0 h (P ≤ 0.05). Data in (A) to (F) are given in nmol/gram of fresh weight [nmol • gFW−1].
(A) Total free fatty acid concentrations in leaves of pxa1 and Col-0 plants exposed to extended dark treatment.
(B) Free fatty acid profile in leaves of pxa1 and Col-0 plants.
(C) Acyl-CoA pool of pxa1 and wild-type leaves.
(D) Acyl-CoA profiles of pxa1 and wild-type leaves.
(E) Quantification of TAG in leaves of pxa1 and Col-0 plants.
(F) Fatty acid profiles of TAG. TAG was recovered from TLC plates and subjected to transmethylation prior to analysis by gas chromatography.
(G) Molecular species of triacylglycerol extracted from pxa1-2 leaves exposed to 20 h of darkness. The sum of the acyl groups are symbolized by the convention, carbon number:number of double bounds. As shown in (F), the fatty acid composition of the TAGs is based almost exclusively on C16 and C18 fatty acids. Therefore, 54:6, for example, indicates a TAG containing two C18 fatty acids and one C16 fatty acid with altogether six double bonds.
- Figure 4.
Impact of Exogenous α-Linolenic Acid on ETR of Isolated Chloroplasts and Plant Growth.
(A) ETR of isolated chloroplasts in response to exogenous α-linolenic acid. Bars represent averages of three independent experiments (n = 6 to 9) ± se.
(B) α-Linolenic acid toxicity in pxa1 and Col-0 plants. Seedlings were germinated and grown on half-strength MS media supplemented with 2% sucrose and transferred to plates containing α-linolenic acid at the concentration indicated after 10 d. Pictures were taken 14 d after transfer.
[See online article for color version of this figure.]
- Figure 5.
Acceleration and Alleviation of the Dark Treatment Phenotype and ATP/ADP ratios in wild-type and pxa1 plants.
(A) Leaf phenotype of 19-d-old adg1-1 plants expressing a PXA1 amiRNA construct grown in continuous light before dark treatment.
(B) Phenotype alleviation through feeding of exogenous sucrose. Three-week-old pxa1-2 and Col-0 plants grown on sterile half-strength MS medium supplemented either with (2%) or without sucrose were exposed to 39 h of darkness and subsequently returned to light for 24 h. pxa1-2 plants grown without exogenous sucrose experienced severe leaf damage and bleaching from this treatment compared with plants grown on sucrose-containing media. This finding was consistent with IMAGING-PAM (pulse amplitude modulation) fluorescence pictures showing either ΦPSII or Fv/Fm in false colors. PAM data were collected before plants were returned to light. Both values were markedly higher in mutants when sucrose was added to the media.
(C) ATP/ADP ratio in leaves of the wild type and pxa1 after different periods of darkness. Average ± se (n = 3 to 4).
(D) Total free fatty acid concentration in leaves of plants grown on half-strength MS medium supplemented with or without sucrose and harvested after 0 and 39 h of darkness. Average ± se (n = 3 to 4).
- Figure 6.
Phototoxicity and PhA Accumulation in pxa1 and kat2 Mutants.
(A) Immediately after 36 h of dark treatment, pxa1 plants (3 weeks old, long-day grown) were partially covered with aluminum foil and illuminated for 24 h. In the uncovered right part of the plant, the phototoxic effect in the leaf becomes obvious, while the covered region maintains a blue-greenish color.
(B) Pigment extraction of 36-h dark-treated plants and control Col-0 (3 weeks old, long-day grown) were spotted on a silica gel matrix and separated with petroleum ether-isopropanol-H2O (100:15:0.25) as running solvent. While extracts of pxa1-2, pxa1-3, or kat2 showed a distinct band in the lower part of the TLC (arrow), this band was clearly absent in dark-treated and untreated Col-0.
(C) and (D) Mass spectra, chemical structure, and UV/VIS spectra of substance from unique band in pxa1 and kat2 extracts. Detected masses and UV/VIS spectrum matches a PhA (C35H36N4O5) standard.
(E) Quantification of PhA in dark-treated Col-0 and pxa1 leaves (3 weeks old, long-day grown). PhA accumulates in pxa1 plants with increasing duration of darkness, whereas levels in Col-0 plants remain close to the detection limit. Bars represent averages of three independent experiments (n = 9) ± se.
[See online article for color version of this figure.]
- Figure 7.
Micrographs of Wild-Type and pxa1 Leaf Tissue at the End of the Light Period and after Extended Darkness.
Electron ([A] to [C] and [G] to [I]) and bright-field ([D] to [F] and [J] to [L]) micrographs of chloroplasts and leaf cross sections prepared from Col-0 wild-type ([A] to [F]) and pxa1-2 ([G] to [L]) leaves at the end of the regular light period (1st column), after 36 h of darkness (2nd column) and after 36 h of darkness plus 4 h of daylight (3rd column). Note the disintegrated structure and increased number of plastoglobules/lipid droplets in chloroplasts of dark-treated pxa1-2 leaves. St, starch granules. Arrowheads indicate plastoglobules. Bars = 1 μm in (A) to (C) and (G) to (I) and 50 μm in (L) (same scale for [D] to [E] and [J] to [L]), respectively.
- Figure 8.
Cartoon Depicting the Suggested Model for Lipid Respiration in Mature Arabidopsis Leaves and Leaves of pxa1 and kat2 Mutants.
In extended darkness, plants respire fatty acids released from the chloroplast for ATP generation in mitochondria via peroxisomal β-oxidation and citrate synthesis. When import of fatty acids into peroxisomes or β-oxidation is impaired, plants accumulate high concentrations of free fatty acids leading to membrane damage, chlorophyll degradation, and other detrimental effects. Provision of an external energy source like sucrose largely prevents the necessity to respire fatty acids and, hence, accumulation of free fatty acids in mutant leaves since ATP can be generated from sucrose.
[See online article for color version of this figure.]
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