Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana

Abstract

Constitutive expression of the cold-regulated COR15a gene of Arabidopsis thaliana results in a significant increase in the survival of isolated protoplasts frozen over the range of -4.5 to -7 degreesC. The increased freezing tolerance is the result of a decreased incidence of freeze-induced lamellar-to-hexagonal II phase transitions that occur in regions where the plasma membrane is brought into close apposition with the chloroplast envelope as a result of freeze-induced dehydration. Moreover, the mature polypeptide encoded by this gene, COR15am, increases the lamellar-to-hexagonal II phase transition temperature of dioleoylphosphatidylethanolamine and promotes formation of the lamellar phase in a lipid mixture composed of the major lipid species that comprise the chloroplast envelope. We propose that COR15am, which is located in the chloroplast stroma, defers freeze-induced formation of the hexagonal II phase to lower temperatures (lower hydrations) by altering the intrinsic curvature of the inner membrane of the chloroplast envelope.

Figure 1

Freezing tolerance of protoplasts isolated from leaves of nonacclimated plants of A. thaliana: RLD (○), the wild type, and T8 (•), which constitutively expresses the COR15a gene. Protoplasts were suspended in either a 0.400 (A and B) or 0.413 (C and D) osmol sorbitol solution before freezing. (A and C) Survival after a conventional freeze/thaw treatment. (B and D) Survival after a freeze/hypertonic-thaw treatment. Survival (percentage of the unfrozen control) was determined by staining with fluorescein diacetate (17). Results shown are the mean and SD of three experiments.

Figure 2

FFEMs of protoplasts isolated from leaves of nonacclimated seedlings (17–20 days old) and suspended in a 0.40 osmol sorbitol solution and frozen to −6°C for 90 min. (A) Protoplasmic fracture face of the plasma membrane (pm) of a T8 protoplast showing a typical, lamellar region, which is characterized by a random distribution of intramembranous particles, melding into a region where the H_II phase has formed. The plasma membrane, which also contains numerous aparticulate domains (apd), is overlaying the chloroplast envelope (ce) with the H_II phase appearing in localized regions. Two other ultrastructural morphologies are commonly observed to be associated with freeze-induced formation of the H_II phase in biological cells: well-ordered striations in the plasma membrane (pm-st) and the chloroplast envelope (ce-st) and loosely ordered swirls. V, vitreous layer of unfrozen suspending medium; i, ice. (Magnification: ×35,700; bar represents 0.5 nm; arrow indicates direction of shadowing). (B) High magnification (×65,200) micrograph of an RLD protoplast illustrating the tightly packed cylinders that are characteristic of the H_II phase (H_II), the well-ordered striations (pm-st), and the loosely ordered swirls (pm-sw). (Bar represents 0.25 nm; arrow indicates direction of shadowing).

Figure 3

³¹P-NMR spectra of DOPE suspended in Pipes buffer in either the absence (A) or the presence (B) of the COR15am polypeptide (2 mg/10 mg DOPE) at temperatures below and above the T_bh. The spectrum for the lamellar phase is characterized by a high-field peak from −2 to −6 ppm and a low-field shoulder from 25 to 35 ppm (e.g., the spectrum at 3.9°C) and the spectrum for the H_II phase is characterized by a low-field peak from 15 to 18 ppm and a high-field shoulder from −3 to −5 ppm (e.g., the spectrum at 10.5°C) (38).

Figure 4

X-ray diffraction measurements of the hexagonal lattice spacing of the H_II phase formed by DOPE in Pipes buffer (20 mM, pH 7.4) in either the absence (○) or the presence (•) of the COR15am polypeptide (2 mg/10 mg DOPE).

Figure 5

³¹P-NMR spectra of a mixture of the major lipid species present in the chloroplast envelope (MGDG:DGDG:SQDG:PC:PG) in proportions (50:30:5:10:5 mol%) that resemble the inner membrane of the chloroplast envelope of rye leaves (27) in either the absence (A) or the presence (B) of the COR15am polypeptide (1 mg/10 mg lipid) at 10°C intervals from 0–40°C.