Chloroplast Membrane Remodeling during Freezing Stress Is Accompanied by Cytoplasmic Acidification Activating SENSITIVE TO FREEZING2

Abstract

Low temperature is a seasonal abiotic stress that restricts native plant ranges and crop distributions. Two types of low-temperature stress can be distinguished: chilling and freezing. Much work has been done on the mechanisms by which chilling is sensed, but relatively little is known about how plants sense freezing. Recently, Arabidopsis (Arabidopsis thaliana) SENSITIVE TO FREEZING2 (SFR2) was identified as a protein that responds in a nontranscriptional manner to freezing. Here, we investigate the cellular conditions that allow SFR2 activation. Using a combination of isolated organelle, whole-tissue, and whole-plant assays, we provide evidence that SFR2 is activated by changes in cytosolic pH and Mg(2+) Manipulation of pH and Mg(2+) in cold-acclimated plants is shown to cause changes similar to those of freezing. We conclude that pH and Mg(2+) are perceived as intracellular cues as part of the sensing mechanism for freezing conditions. This evidence provides a specific molecular mechanism to combat freezing.

Figure 1.

SFR2 is posttranslationally activated by pH and magnesium ions. Wild-type Arabidopsis plants were cold acclimated at 6°C for 1 week, incubated overnight at the temperatures indicated at top, and then sampled for lipids and proteins. A, Immunoblot detecting SFR2 protein levels. B, Thin-layer chromatogram separating lipids identified at right visualized with a sugar-specific stain. Images shown are representative of three separate plant growth trials. C to G, Isolated chloroplasts were incubated with radiolabeled UDP-Gal in 300 mm sorbitol, 50 mm HEPES, pH 7.5, or modified buffers as indicated below the graph axis. Radiolabel in the oligogalactolipid product TGDG is quantified as a percentage of total radiolabeled lipids. Error bars represent sd of at least three separately grown trials. Asterisks represents significance (P ≤ 0.05) between the treatment and the condition most closely mimicking normal cytoplasm (300 mm sorbitol, pH 7.4, 0 mm hydrogen peroxide [H₂O₂], 0 mm cumene hydroperoxide, no divalent cations [F], or 0.4 mm MgCl₂ [G]).

Figure 2.

pH and magnesium changes activate SFR in whole tissues. Thin-layer chromatograms show the separation of lipids from extracts of Arabidopsis (A.t.) shoots or pea (P.s.) leaves floated on 20 mm of the acid indicted at left adjusted to the pH indicated at top with dipotassium phosphate for 1 h. 7M indicates pH 7 with an additional 20 mm MgCl₂. TGDG is indicated by arrowheads. Images shown are representative of three separate plant growth trials.

Figure 3.

Cytosolic pH changes during freezing and acetic acid treatment. A, Arabidopsis plants stably transformed with PtGFP were grown under control conditions (22°C), or grown and cold acclimated at 6°C for 1 week, or cold acclimated and frozen overnight at −6°C. A cold stage (4°C) was used to measure chilled plants. Ratiometric fluorescence was measured in hypocotyls, with excitation at 488 nm divided by excitation at 405 nm with detection constantly between 505 and 530 nm. Bars = 22 µm. B, Pure PtGFP protein was measured identically to A in microcapillaries at 22°C or on the cold stage (4°C) to provide a pH scale. C, Ratiometric fluorescence images of two independent lines of PtGFP, including those shown in A, were transformed into pH as described in “Materials and Methods” and are graphed according to most recently exposed temperature. Statistical significance values are as follows: 22°C versus 6°C (all samples), P = 0.0325; 6°C versus −6°C (all samples), P = 5 × 10⁻⁸; for line 1 individually: 22°C versus 6°C, P = 0.215; 6°C versus −6°C, P = 0.0006; for line 2 individually: 22°C versus 6°C, P = 0.0661; 6°C versus −6°C, P = 9 × 10⁻⁸. D, The same two independent lines of PtGFP used in C and A were untreated or floated on water or 20 mm acetic acid at pH 5 for 1 h, mimicking treatments in Figure 2. Statistical significance values are as follows: acetic acid versus water (all samples), P = 1.21 × 10⁻¹⁶; acetic acid versus untreated (all samples), P = 1.3 × 10⁻²⁴; water versus untreated (all samples), P = 1.09 × 10⁻⁹; for line 1 individually: acetic acid versus water, P = 0.0052; acetic acid versus untreated, P = 2.5 × 10⁻¹⁰; water versus untreated, P = 4.44 × 10⁻¹⁰; for line 2 individually: acetic acid versus water, P = 5.22 × 10⁻¹⁹; acetic acid versus untreated, P = 2.54 × 10⁻¹⁶; water versus untreated, P = 0.0023.

Figure 4.

SFR2 is not substrate limited and does not stably interact with other proteins. A, Ten-day-old wild-type (WT) or sfr2 Arabidopsis plants were transferred to regular medium or medium lacking phosphate for 10 d, and then lipids were extracted. Resulting lipids were analyzed by thin-layer chromatography for the presence of TGDG (black arrowhead). The location of digalactosyldiacylglycerol (DGDG) is indicated by the white arrowhead. B, Immunoblot of 40 µg of chlorophyll equivalent wild-type (top) or sfr2 (bottom) chloroplasts solubilized with 2% digitonin separated in two dimensions, 4% to 14% blue-native PAGE in the first dimension and 7.5% denaturing PAGE in the second dimension, detected with the SFR2 antiserum. Arrowheads indicate SFR2-specific signal, while asterisks identify nonspecific signal. C, Comparisons of SFR2 leaf protein two-dimensional immunoblots of plants grown at 22°C, cold acclimated for 1 week (6°C) or cold acclimated and frozen overnight at −6°C. D, Comparisons of SFR2 two-dimensional immunoblots as in B for mutants and controls identified at left. E, Wild-type or mutant Arabidopsis as indicated at top were tested for the ability to produce TGDG (arrowhead) in response to a 1-h incubation in 20 mm acetic acid, pH 5, or to withstand freezing at −6°C (bottom). All portions of the figure are representative of at least three separately grown biological replicates.

Figure 5.

pH and Mg²⁺ treatments mimic lipid changes due to freezing. A to D, Plants were grown at 22°C for 3 weeks and cold acclimated at 6°C for 1 week for all treatments (Cold). They were subsequently frozen at −6°C overnight (Frozen) or floated on 20 mm acetic acid, pH 5 (AcOH), 20 mm acetic acid, pH 5, with 10 mm magnesium chloride (AcOH + Mg), or water for 3 h. All plants were sampled as rosettes with roots removed. Molar percentage (A and C) of MGDG, PG, and TAG relative to total lipid amount and fatty acid profiles (B and D) of each lipid species relative to total fatty acids for each individual fatty acid were quantified. Values are biological replicate means ± sd. Each biological replicate consists of an average of three or four technical replicates. E, Lipid droplets were visualized with confocal microscopy after Nile Red staining and quantified as the number of lipid droplets per cell. Boxes encompass the interquartile range, with the central line representing the median. Whiskers represent maximum and minimum counts. For all data, significance (P ≤ 0.05) between control and treatment is represented by double daggers, and asterisks represent significance (P ≤ 0.05) between the wild type (WT) and sfr2.