The sensitive to freezing3 mutation of Arabidopsis thaliana is a cold-sensitive allele of homomeric acetyl-CoA carboxylase that results in cold-induced cuticle deficiencies

Abstract

The sfr3 mutation causes freezing sensitivity in Arabidopsis thaliana. Mapping, sequencing, and transgenic complementation showed sfr3 to be a missense mutation in ACC1, an essential gene encoding homomeric (multifunctional) acetyl-CoA carboxylase. Cuticle permeability was compromised in the sfr3 mutant when plants were grown in the cold but not in the warm. Wax deposition on the inflorescence stem of cold-grown sfr3 plants was inhibited and the long-chain components of their leaf cuticular wax were reduced compared with wild-type plants. Thus, freezing sensitivity of sfr3 appears, from these results, to be due to cuticular deficiencies that develop during cold acclimation. These observations demonstrated the essential role of the cuticle in tolerance to freezing and drought.

Fig. 1.

The sfr3 phenotype. (A) Comparison of wild-type (left) and sfr3 (right) plants after a freezing test. Cold acclimated plants were frozen at –6.0 °C for 16h and returned to pre-acclimation growth conditions for recovery. The picture was taken after 4 days recovery. All leaves of the wild-type appeared healthy and green, as did the older, fully expanded leaves of sfr3 plants. The younger leaves of sfr3 plants showed severe damage and were discoloured and collapsed. (B) Wild-type (left) and sfr3 (right) plants after water was withheld for 25 days from plants maintained at 4 °C. Damage was apparent across the whole rosette but was most severe in young leaves (none fully expanded).

Fig. 2.

Location of the sfr3 mutation in the ACC1 gene. (A) Schematic representation of the ACC1 gene showing the location of the sfr3 point mutation. Solid boxes represent exons. (B) Amino acid change in the conceptual translation product of the ACC1 gene. The hatched box, dotted box, and solid box represent the biotin carboxylase domain, biotin carboxyl carrier domain and carboxyl transferase domain, respectively. (C) Alignment of amino acids in the conserved region of the biotin carboxylase domain of acetyl-CoA carboxylases of various species. Bold letters indicate invariant amino acids while the asterisk identifies the amino acid changed in the sfr3 mutant. AtACC1, Arabidopsis thaliana ACC1 (At1g36160); TaACC, Triticum aestivum cytosolic ACCase (A57710); ZmACC, Zea mays ACCase (T02235); DmACC, Drosophila melanogaster ACCase (CG11198-PB); HsACC, Homo sapiens ACCase (S41121); ScACC, Saccharomyces cerevisiae ACCase (P11029).

Fig. 3.

Toluidine blue staining of sfr3 and wild-type seedlings grown in the cold for 10 d. A control wild-type plant before (A) and after (E, I) staining is shown. Three different sfr3 plants are shown before (B–D) and after (F–H and J–L) staining. The images in the lower panel (I–L) are close ups of the images in the panel above to show details of the different staining patterns.

Fig. 4.

Graphs showing the rate of leaching of chlorophyll into ethanol from sfr3 plants compared with wild-type plants. The results from warm-grown plants (A) and warm-grown plants transferred to the cold for 24h (B), 4 d (C), and 10 d (D) are shown. Experiments were performed in triplicate and results are shown as means ± standard deviation. The total amount of chlorophyll present in the leaves of sfr3 plants, measured by total extraction of chlorophyll in 96% ethanol, was not significantly different from that of wild-type plants (data not shown).

Fig. 5.

Scanning electron microscopy images showing wax crystals on the inflorescence stem of wild-type (A, C) and sfr3 (B, D) plants. The surface of wild-type (A) and sfr3 (B) stems that developed in the warm before transfer to 4 °C for 21 d are decorated by densely distributed columnar-shaped epicuticular wax crystals. Wild-type stems that had initiated bolting in the warm before transfer to the cold showed a dense decoration of wax crystals (C). sfr3 stems that were grown in the same way (initiation in the warm and growth in the cold) showed a total lack of visible wax crystals (D). Bars, 10 µm.

Fig. 6.

Graph showing a comparison of the relative amounts of the major classes of leaf wax components isolated from warm-grown and cold-treated sfr3 plants. The ratio of each wax component relative to the amount on wild-type plants grown under the same conditions (warm or cold) is shown. Chemical classes and chain lengths are labelled on the horizontal axis. Statistically significant differences (Student’s t-test, P <0.05) between wild-type and sfr3 plants treated in the same way are indicated by an asterisk.

Fig. 7.

Image showing the effects of long-term growth at 4 °C on inflorescence development in sfr3 plants. Warm-grown 4-week-old plants were transferred to 4 °C growth conditions shortly after the inflorescence stem had initiated and maintained at this temperature for 10 weeks. Wild-type plants (A, left plant) developed normal inflorescence stems supporting flowers and siliques, while sfr3 plants developed only stunted inflorescence stems (A, right plant). Flowers of normal appearance developed on wild-type plants (B), while those on sfr3 plants were abnormal (C). Petals were shrunken and brown and did not expand, and no siliques were observed.